U.S. patent application number 11/584751 was filed with the patent office on 2008-02-07 for method for small volume nucleic acid synthesis.
Invention is credited to James M. Kadushin.
Application Number | 20080033159 11/584751 |
Document ID | / |
Family ID | 39030071 |
Filed Date | 2008-02-07 |
United States Patent
Application |
20080033159 |
Kind Code |
A1 |
Kadushin; James M. |
February 7, 2008 |
Method for small volume nucleic acid synthesis
Abstract
A method for synthesis of nucleic acids, with particular use for
synthesis of cDNA. The method allows application of the cDNA to a
microarray without the need for concentration or purification of
the cDNA post-cDNA synthesis.
Inventors: |
Kadushin; James M.;
(Gilbertsville, PA) |
Correspondence
Address: |
LAW OFFICE OF MORRIS E. COHEN
1122 CONEY ISLAND AVENUE
SUITE 217
BROOKLYN
NY
11230
US
|
Family ID: |
39030071 |
Appl. No.: |
11/584751 |
Filed: |
October 20, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11388772 |
Mar 24, 2006 |
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11584751 |
Oct 20, 2006 |
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PCT/US04/31804 |
Sep 27, 2004 |
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11388772 |
Mar 24, 2006 |
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10825776 |
Apr 16, 2004 |
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11388772 |
Mar 24, 2006 |
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10050088 |
Jan 14, 2002 |
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10825776 |
Apr 16, 2004 |
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10234069 |
Sep 3, 2002 |
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PCT/US04/31804 |
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PCT/US03/09232 |
Mar 25, 2003 |
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PCT/US04/31804 |
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60506247 |
Sep 26, 2003 |
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60261231 |
Jan 13, 2001 |
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60316116 |
Aug 31, 2001 |
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60367438 |
Mar 25, 2002 |
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Current U.S.
Class: |
536/25.32 |
Current CPC
Class: |
C12N 15/1096
20130101 |
Class at
Publication: |
536/025.32 |
International
Class: |
C07H 21/00 20060101
C07H021/00 |
Claims
1. A method comprising: taking an initial sample of RNA; reverse
transcribing the sample of RNA to synthesize a sample of cDNA; and,
applying the sample of cDNA to a microarray; wherein the sample of
cDNA is applied to the microarray without concentration of the
sample of cDNA after the synthesis of the cDNA and before
application of the sample of cDNA to the microarray.
2. A method as claimed in claim 1, wherein the sample of cDNA is
applied to the microarray without purification of the sample of
cDNA after synthesis of the sample of cDNA and before application
of the sample of cDNA to the microarray.
3. A method as claimed in claim 1, wherein the initial sample of
RNA comprises total RNA.
4. A method as claimed in claim 1, wherein the initial sample of
RNA comprises messenger RNA.
5. A method as claimed in claim 1, wherein the initial sample of
RNA comprises from approximately 0.1 to 1 micrograms of total
RNA.
6. A method as claimed in claim 1, wherein the initial sample of
RNA comprises from approximately 1 to 1000 nanograms of mRNA.
7. A method as claimed in claim 1, further comprising the step of
hybridizing a dendrimer to the cDNA in the sample of cDNA.
8. A method as claimed in claim 1, further comprising the step of
hybridizing a dendrimer to the cDNA in the sample of cDNA, wherein
the dendrimer is labeled to produce a detectable signal.
9. A method as claimed in claim 1, further comprising the step of
hybridizing a dendrimer to the cDNA in the sample of cDNA, wherein
the dendrimer is labeled to produce a detectable signal, and
wherein the dendrimer is labeled with greater than 500 fluorescent
dyes on each dendrimer molecule.
10. A method comprising: taking an initial sample of RNA; reverse
transcribing the sample of RNA to synthesize a sample of cDNA;
applying the sample of cDNA to a microarray, wherein the microarray
has nucleic acid samples affixed thereto; allowing the cDNA in the
sample of cDNA to hybridize to the nucleic acid samples on the
microarray; and, hybridizing a dendrimer to the cDNA in the sample
of cDNA; wherein the sample of cDNA is applied to the microarray
without concentration of the sample of cDNA after the synthesis of
the cDNA and before application of the sample of cDNA to the
microarray.
11. A method as claimed in claim 10, wherein the sample of cDNA is
applied to the microarray without purification of the sample of
cDNA after synthesis of the sample of cDNA and before application
of the sample of cDNA to the microarray.
12. A method as claimed in claim 10, wherein the initial sample of
RNA comprises total RNA.
13. A method as claimed in claim 10, wherein the initial sample of
RNA comprises messenger RNA.
14. A method as claimed in claim 10, wherein the initial sample of
RNA comprises from approximately 0.1 to 1 micrograms of total
RNA.
15. A method as claimed in claim 10, wherein the initial sample of
RNA comprises from approximately 1 to 1000 nanograms of mRNA.
16. A method as claimed in claim 10, further comprising the step of
hybridizing a dendrimer to the cDNA in the sample of cDNA, wherein
the dendrimer is labeled to produce a detectable signal.
17. A method as claimed in claim 10, further comprising the step of
hybridizing a dendrimer to the cDNA in the sample of cDNA, wherein
the dendrimer is labeled to produce a detectable signal, and
wherein the dendrimer is labeled with greater than 500 fluorescent
dyes on each dendrimer molecule.
Description
RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
Nonprovisional application Ser. No. 11/388,772 filed Mar. 24, 2006,
which is a continuation of PCT Application Serial No.
PCT/US2004/031804 ("the '804 application") filed Sep. 27, 2004,
which claims the priority of U.S. Provisional Application Ser. No.
60/506,247 filed Sep. 26, 2003.
[0002] The '804 application is also a continuation-in-part of U.S.
Nonprovisional application Ser. No. 10/825,776 filed Apr. 16, 2004
(pending), which is a continuation of U.S. Nonprovisional
application Ser. No. 10/050,088 filed Jan. 14, 2002 (abandoned),
which claims the priority of U.S. Provisional Application Ser. No.
60/261,231 filed Jan. 13, 2001.
[0003] The '804 application is also a continuation-in-part of U.S.
Nonprovisional application Ser. No. 10/234,069 filed Sep. 3, 2002
(pending), which claims the priority of U.S. Provisional
Application Ser. No. 60/316,116 filed Aug. 31, 2001.
[0004] The '804 application is also a continuation-in-part of PCT
Application Serial No. PCT/US03/09232 filed Mar. 25, 2003 (pending)
("the '232 application") which claims the priority of U.S.
Provisional Application Ser. No. 60/367,438 filed Mar. 25,
2002.
[0005] The priority of all of those applications is claimed, all of
which are fully incorporated herein by reference.
FIELD OF THE INVENTION
[0006] The present invention relates to an improved method for
nucleic acid synthesis.
BACKGROUND OF THE INVENTION
[0007] The synthesis of nucleic acids is one of the cornerstones of
modern molecular biology, and is utilized in a large variety of
experimental and diagnostic techniques. Of the various forms of
nucleic acids currently known, cDNA or "Complementary DNA" is a DNA
copy made off of a messenger RNA (mRNA) or other type of RNA
molecule template.
[0008] Synthesis of the cDNA molecule from the original mRNA is
accomplished using an enzyme known as Reverse Transcriptase (RT),
which is an RNA dependent DNA polymerase. Reverse Transcriptase was
initially discovered in connection with retroviruses, and can be
obtained via purification from such a virus, such as avian
myoblastosis virus (AMV) or Moloney murine leukemia virus (M-MuLV),
or from a cell containing the cloned gene.
[0009] Using the mRNA isolated from a given cellular source, a
library can be constructed of cDNA molecules complementary to the
cellular mRNA. That cDNA library can then be used for various
experimental purposes. For example, a cDNA library formed from a
particular tissue type can used for gene expression analysis, i.e.
to provide information regarding the expression of nucleic acids in
the initial sample. Gene expression analysis may be of use in a
variety of applications, including, for example, the identification
of novel expression of genes, the correlation of gene expression to
a particular phenotype, screening for disease predisposition, and
identifying the effect of a particular agent on cellular gene
expression, such as in toxicity testing and screening for new drug
compounds.
[0010] To perform an analysis, total (or messenger) RNA is
extracted from the desired sample of cells. Copies of complementary
DNA (cDNA) are generated from the RNA through reverse
transcription. The cDNA copies are tagged with a marker or label
such as a fluorescent marker and broken up into short
fragments.
[0011] As discussed in prior patent applications of the present
inventor, analysis of the cDNA sequences in a given sample can be
particularly efficient when conducted using high-speed technologies
for nucleic acid analysis such as a DNA microarray (see e.g. U.S.
Nonprovisional application Ser. No. 10/234,069 filed Sep. 3, 2002
which claims the priority of U.S. Provisional Application Ser. No.
60/316,116 filed Aug. 31, 2001, and "Methods for Blocking
Nonspecific Hybridizations of Nucleic Acids", International
Application No. PCT/US02/027799 filed 3 Sep. 2002, International
Publication No. WO 03/020902 A2, all of which are fully
incorporated herein by reference). All microarrays operate on a
similar principle: a substantially planar substrate such as a glass
slide or a silicon chip or nylon membrane is coated with a grid of
tiny spots of about 20 to 100 microns in diameter. Each spot (i.e.
feature) contains millions of copies of a short sequence of DNA or
nucleotides; a computer keeps track of the location of each
sequence on the substrate, allowing the user to conduct thousands
of miniature test-tube like reactions simultaneously.
[0012] After tagging of the cDNA copies, the tagged fragments are
washed over the microarray and left overnight, to allow the tagged
fragments to hybridize with the DNA attached to the microarray.
After hybridization, the features on the microarray that have
paired with the fluorescent cDNA emit a fluorescent signal that can
be viewed with a microscope or detected by a computer. In this
manner, one can learn which sequences on the microarray match the
cDNA of the test sample. Although there are occasional mismatches,
the employment of millions of probes in each spot or feature ensure
fluorescence is detected only if the complementary cDNA is present.
The more intense the fluorescent signal, (i.e. the brighter the
spot) the more matching cDNA was present in the cell.
[0013] Unfortunately, however, in the traditional methods known in
the art, after preparation of the cDNA through reverse
transcription, concentration of the cDNA must be conducted before
the cDNA can be used in the hybridization mix applied to the
microarray. Often purification must be conducted as well, such as
when the cDNA has been labelled using dyes. These concentration
and/or purification steps add additional time and expense to the
experimental analysis.
[0014] One typical method for nucleic acid concentration, for
example, is ethanol precipitation. An illustrative protocol for
ethanol precipitation to enable cDNA concentration is as
follows:
Ethanol Precipitation of Synthesized cDNA
[0015] 1. Adjust the volume of synthesized cDNA to 130 uL with
1.times.TE buffer. [0016] 2. Add 3 .mu.l of the linear acrylamide
(5.0 mg/mL) to the synthesized cDNA mix. [0017] 3. Add 6 .mu.l of
5M NaCl or 250 .mu.l 3M Ammonium Acetate and mix. [0018] 4. Add 540
.mu.l of 100% ethanol if using NaCl or 875 .mu.l of 100% ethanol if
using 3M Ammonium Acetate. Mix by moderate vortexing. [0019] 5.
Incubate at -20 degrees Celsius for 30 minutes. [0020] 6.
Centrifuge the sample at >10,000 g for 15 minutes. [0021] 7.
Carefully aspirate the supernatant to avoid loss of the cDNA
pellet. Do not decant, as decanting may dislodge the pellet and
cause it to be lost. [0022] 8. Add 300 .mu.l of 70% ethanol to the
cDNA pellet. Gently mix by tapping the side of the tube. Avoid
overmixing, which may cause the cDNA pellet to break up. [0023] 9.
Centrifuge at >10,000 g for 5 minutes and remove the
supernatant. Do not decant. [0024] 10. Dry the cDNA pellet
completely by heating for 10-30 minutes at 65 degrees Celsius. If
the cDNA pellet is not completely dry, it will be difficult to
resuspend, and incomplete resuspension may produce high speckled
background on the microarray and/or weak results. [0025] 11.
Proceed to hybridization of the cDNA to the array.
[0026] Although ethanol precipitation is a traditional and accepted
method for nucleic acid concentration, unfortunately, it may lead
to variable results due to partial or complete loss of the pelleted
cDNA or incomplete re-solubilization of the precipitated cDNA. For
example, as can be seen above, problems may occur if the ethanol
precipitation procedure is not performed carefully because reverse
transcription of small quantities of RNA produces a cDNA pellet
that is very small and easily lost during processing or by
adherance to the inside of pipet tips.
[0027] An alternate method for concentration of the cDNA pellet is
concentration with Microcon.RTM. microconcentrators. A sample
protocol for Microcon.RTM. concentration is as follows:
Concentration of cDNA with Millipore Microcon.RTM. YM-30
Centrifugal Filter Devices
[0028] cDNA samples may be concentrated using the Millipore
Microcon.RTM. YM-30 Centrifugal Filter Devices (30,000 molecular
weight cutoff, Millipore catalog number 42409). The following
protocol is an example of a method provided to reduce the volume of
the cDNA synthesis reaction from 130 .mu.l to 3-10 .mu.l, for
hybridization to an array. (Note: while the following sample
protocol is similar to that provided by the manufacturer, it
includes minor modifications for use with the 3DNA Array 350 kit.
Additionally, users of the Microcon YM-30 should evaluate their own
centrifuge settings to determine the optimal time and speed
settings to yield final volumes of 3-10 .mu.l). [0029] 1. Place the
Microcon.RTM. YM-30 sample reservoir into the 1.5 ml collection
tube. [0030] 2. Pre-wash the reservoir membrane by adding 100 .mu.l
TE pH 8.0 to the Microcon.RTM. YM-30 sample reservoir. [0031] 3.
Place the tube/sample reservoir assembly into a fixed angle rotor
tabletop centrifuge capable of 10-14,000 g. [0032] 4. Spin for 3
minutes at 10-14,000 g. [0033] 5. Add all 130 .mu.l from the cDNA
reaction to the Microcon.RTM. YM-30 sample reservoir. Do not touch
the membrane with the pipet tip. [0034] 6. Place the tube/sample
reservoir assembly into a fixed angle rotor tabletop centrifuge
capable of 10-14,000 g. [0035] 7. Centrifuge for 8-10 minutes at
10-14,000 g. [0036] 8. Remove the tube/sample reservoir assembly.
Separate the collection tube from the sample reservoir with care,
avoiding spilling any liquid in the sample reservoir. [0037] 9. Add
5 .mu.l of 1.times.TE buffer (10 mM Tris-HCl, pH 8.0/1 mM EDTA) to
the sample reservoir membrane without touching the membrane. Gently
tap the side of the concentrator to promote mixing of the
concentrate with the 1.times.TE buffer. [0038] 10. Carefully place
the sample reservoir upside down on a new collection tube.
Centrifuge for 2 minutes at top speed in the same centrifuge.
[0039] 11. Separate the sample reservoir from the collection tube
and discard the reservoir. Note the volume collected in the bottom
of the tube (3-10 .mu.l total volume). The cDNA sample may be
stored in the collection tube for later use. [0040] 12. Proceed to
hybridization of the cDNA to the array.
[0041] As can be seen from the above examples, the sample
concentration protocols traditionally utilized prior to application
of the cDNA to a microarray require a time consuming extra series
of steps after cDNA synthesis. In some cases, these additional
steps can decrease performance and the results obtained in the
assay.
[0042] Accordingly, it is an object of the present invention to
provide an improved method for cDNA synthesis which eliminates the
need for the post-synthesis sample concentration discussed
above.
SUMMARY OF THE INVENTION
[0043] It is an object of the present invention to provide an
improved method for nucleic acid synthesis.
[0044] It is a further object of the present invention to provide
an improved method for synthesis of cDNA.
[0045] It is a further object of the present invention to provide a
method which avoids the need for concentration of the cDNA after
cDNA synthesis.
[0046] It is a further object of the present invention to provide a
method which avoids the need for a purification step to purify
undesirable molecules from the cDNA sample after cDNA
synthesis.
[0047] It is a further object of the present invention to provide a
method for synthesis of cDNA using very small quantities of sample
materials.
[0048] It is a further object of the present invention to provide a
method for synthesis of cDNA which provides increased
sensitivity.
[0049] It is a further object of the invention to provide an
improved method for cDNA synthesis for use with microarrays.
[0050] It is a further object of the present invention to provide a
method for synthesis of cDNA, which allows application of the cDNA
to a microarray without the need for concentration of the cDNA
after cDNA synthesis.
[0051] It is a further object of the present invention to provide a
method for synthesis of cDNA, which allows application of the cDNA
to a microarray without the need for purification of undesirable
molecules from the cDNA sample after cDNA synthesis.
[0052] It is a further object of the present invention to provide a
method for synthesis of cDNA for application to microarrays in a
readily automatable fashion.
[0053] Further to the above objects, in accordance with the present
invention, a method is disclosed for improved nucleic acid
synthesis. The method provides higher sensitivity results from very
small quantities of sample materials than provided by traditional
methods in the art by eliminating a sample concentration protocol
after nucleic acid synthesis.
[0054] In the preferred embodiments, the invention is used for the
synthesis of cDNA (complementary DNA) from an initial RNA samples,
such as mRNA from a source of interest. In further preferred
embodiments, the method is used for nucleic acids intended for
application to microarrays, for assay of those nucleic acids using
hybridization of the synthesized nucleic acids to known molecules
affixed to the array. In further preferred embodiments, the assays
are conducted using dendritic nucleic acid reagents. Yet further
preferably, capture sequences are used to label the synthesized
nucleic acids and/or the nucleic acids on the array.
[0055] In further embodiments of the invention, kits may be
provided for conducting the methods disclosed herein. Additionally,
the processes can also be used for other types of sample
preparations for unrelated applications.
[0056] The avoidance of the need for a post-synthesis sample
concentration protocol is a significant advantage of the present
method, particularly since such protocols can cause excessive loss
of sample. The method is particularly useful with small quantity
preparations starting with less than one microgram (1000 nanograms)
of total RNA or equivalent nucleic acid sample.
[0057] A further significant advantage of the present invention is
that it provides a reduction of the time and number of operations
required to perform complete cDNA synthesis. This advantage is of
particular importance for a variety of contexts and applications,
including the needs of research laboratories, diagnostic kits,
clinical settings, and so forth.
[0058] Yet a further significant advantage of the invention is that
it provides a better consistency of final cDNA yield based on the
same input materials, leading to better reproducibility of
results.
[0059] Further objects and advantages of the invention will become
apparent in conjunction with the detailed disclosure provided
herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0060] FIG. 1 is a schematic view of a preferred method in
accordance with the present invention.
DETAILED DESCRIPTION OF THE INVENTION AND THE PREFERRED
EMBODIMENTS
[0061] Before the present invention is further described, it is to
be understood that the invention is not limited to the particular
embodiments of the invention described herein, as variations of the
particular embodiments may be made and still fall within the scope
of the invention or the appended claims. It is also to be
understood that the terminology employed is for the purpose of
describing particular embodiments, and is not intended to be
limiting.
[0062] The present invention is generally directed to a method for
provided for nucleic acid synthesis which avoids the need for
post-synthesis sample concentration. The cost effective and
efficient manner by which the nucleic acid sequence samples are
prepared and by which the methods of the present invention are
implemented, using conventional laboratory techniques, equipment
and reagents, make them especially suitable for research and
clinical use, and for automation.
[0063] The invention is particularly suitable for nucleic acid
synthesis, such as cDNA synthesis, conducted in conjunction with
assay on a microarray. In such methods, an array of DNA or gene
probes fixed or stably associated with the surface of a
substantially planar substrate ("a microarray") is contacted with a
sample of target nucleic acids under hybridization conditions
sufficient to produce a hybridization pattern of complementary
probe/target complexes. A variety of different microarrays which
may be used are known in the art. The hybridized samples of nucleic
acids are then targeted by labeled probes and hybridized to produce
a detectable signal corresponding to a particular hybridization
pattern. The individual labeled probes hybridized to the target
nucleic acids are all capable of generating the same signal of
known intensity. Thus, each positive signal in the microarray can
be "counted" in order to obtain quantitative information about the
genetic profile of the target nucleic acid sample.
[0064] The DNA or gene probes of the microarrays which are capable
of sequence specific hybridization with target nucleic acid may be
polynucleotides or hybridizing analogues or mimetics thereof,
including, but not limited to, nucleic acids in which the
phosphodiester linkage has been replaced with a substitute linkage
group, such as phophorothioate, methylimino, methylphosphonate,
phosphoramidate, guanidine and the like, nucleic acids in which the
ribose subunit has been substituted, e.g. hexose phosphodiester;
peptide nucleic acids, and the like. The length of the probes will
generally range from 10 to 1000 nucleotides, although the present
invention is not limited to probes of such lengths. In some
embodiments of the invention, for example, the probes will be
oligonucleotides having from 15 to 150 nucleotides and more usually
from 15 to 100 nucleotides. In other embodiments the probes will be
longer, usually ranging in length from 150 to 1000 nucleotides,
where the polynucleotide probes may be single or double stranded,
usually single stranded, and may be PCR fragments amplified from
cDNA. The DNA or gene probes on the surface of the substrates will
preferably correspond to known genes of the physiological source
being analyzed and be positioned on the microarray at a known
location so that positive hybridization events may be correlated to
expression of a particular gene in the physiological source from
which the target nucleic acid sample is derived. Because of the
manner in which the target nucleic acid sample is generated, as
described below, the microarrays of gene probes will generally have
sequences that are complementary to the non-template strands of the
gene to which they correspond.
[0065] The substrates with which the gene probes are stably
associated may be fabricated from a variety of materials, including
plastic, ceramic, metal, gel, membrane, glass, and the like. The
microarrays may be produced according to any convenient and
conventional methodology, such as preforming the gene probes and
then stably associating them with the surface of the support or
growing the gene probes directly on the support. A number of
different microarray configurations and methods for their
production are known to those of skill in the art, one of which is
described in Science, 283, 83, 1999, the content of which is
incorporated herein by reference.
[0066] In accordance with the method of the present invention, a
desired microarray is provided having the probe nucleic acid
sequences stably affixed thereto. In addition, a sample is provided
having the target molecules of interest for study. The target
molecules are labelled for detection, the term "label" is used
herein to refer to agents that are capable of providing a
detectable signal, either directly or through interaction with one
or more additional members of a signal producing system. The label
is one which preferably does not provide a variable signal, but
instead provides a constant and reproducible signal over a given
period of time. The target molecules can be labelled prior to or
after application of target to the array, although prior labelling
is generally preferred. Preferably a very sensitive signal
generating method is used, such as a dendrimer or another
comparably sensitive signal generating methods, e.g. relative light
scatter detection using nanogold labels, such as those of Genicon
Inc./Invitrogen.
[0067] In conjunction with the present inventions, dendritic
nucleic acid molecules are particularly preferred for their
detection capabilities (although any type of labelled molecules of
suitable sensitivity can be utilized with the inventions disclosed
herein). Dendritic nucleic acid molecules, or dendrimers are
complex, highly branched molecules, comprised of a plurality of
interconnected natural or synthetic monomeric subunits of
double-stranded DNA. Dendrimers are described in greater detail in
Nilsen et al., Dendritic Nucleic Acid Structures, J. Theor. Biol.,
187, 273-284 (1997); in Stears et al., A Novel, Sensitive Detection
System for High-Density Microarrays Using Dendrimer Technology,
Physiol. Genomics, 3: 93-99 (2000); and in various U.S. patents,
such as U.S. Pat. Nos. 5,175,270; 5,484,904; 5,487,973; 6,072,043;
6,110,687; and 6,117,631; all of which are fully incorporated
herein by reference. Likewise, various inventions relating to
dendrimers and their use on microarrays are described in PCT
Application Serial No. PCT/US03/09232 filed 25 Mar. 2003, U.S.
Provisional Application Ser. No. 60/367,438 filed Mar. 25, 2002,
U.S. Nonprovisional application Ser. No. 10/825,776 filed Apr. 16,
2004, U.S. Nonprovisional application Ser. No. 10/050,088 filed
Jan. 14, 2002; U.S. Provisional Application Ser. No. 60/261,231
filed Jan. 13, 2001; U.S. Nonprovisional application Ser. No.
10/730,823 filed Dec. 8, 2003; U.S. Nonprovisional application Ser.
No. 10/393,519 filed Mar. 20, 2003; PCT Application Serial No.
PCT/US01/29589 filed Sep. 20, 2001; U.S. Provisional Application
Ser. No. 60/234,060 filed Sep. 20, 2000; PCT Application Serial No.
PCT/US01/29589 filed Sep. 20, 2001; U.S. Nonprovisional application
Ser. No. 09/908,950 filed Jul. 19, 2001; U.S. Provisional
Application Ser. No. 60/219,397, filed Jul. 19, 2000; U.S.
Provisional Application Ser. No. 60/187,681 filed Mar. 8, 2000;
U.S. Nonprovisional application Ser. No. 09/802,162 filed Mar. 8,
2001; and U.S. Provisional Application Ser. No. 60/187,681 filed
Mar. 8, 2000; PCT Application Serial No. PCT/US2003/009232; and PCT
Application No. PCT/US2003/025865; all of which are fully
incorporated herein by reference.
[0068] Dendrimers comprise two types of single-stranded
hybridization "arms" on the surface which are used to attach two
key functionalities. A single dendrimer molecule may have at least
one hundred arms of each type on the surface. One type of arm is
used for attachment of a specific targeting molecule to establish
target specificity and the other is used for attachment of a label
or marker. The molecules that determine the target and labeling
specificities of the dendrimer are attached either as
oligonucleotides or as oligonucleotide conjugates. Using simple DNA
labeling, hybridization, and ligation reactions, a dendrimer
molecule may be configured to act as a highly labeled, target
specific probe.
[0069] The prepared mixture is formulated in the presence of a
suitable buffer to yield a dendrimer hybridization mixture
containing dendrimers with fluorescent labels attached to one type
of "arm", and with oligonucleotides attached to another type of
"arm", complementary to the capture sequences of the RT primer
bound cDNA fragments. An oligonucleotide designed to block
non-specific interaction of the cDNA or the dendrimer to the
nucleic acid spotted on the array surface can also be added at this
time; blocking oligonucleotides containing the multiplicities of
the same nucleic acid base may be used for blocking long stretches
of the same complementary base found on the cDNA derived from the
RNA sample and the nucleic acid probes on the microarray
surface.
[0070] To prepare fluorescent labeled dendrimer, the complementary
sequences to the capture sequence on the Cy3.RTM. RT primer and the
Cy5.RTM. RT primer are ligated, separately, to the purified
dendritic core material as prepared by the previously described
methods (see Nilson et al., supra, and U.S. Pat. Nos. '270, '904,
and '973, supra.). Thirty nucleotide long oligonucleotides
complementary to the outer arms of a four-layer dendrimer having a
5' Cy3.RTM. or Cy5.RTM. are then synthesized. (Oligos etc., Inc.,
Wilsonville, Oreg.). The Cy3.RTM. and Cy5.RTM. oligonucleotides are
then hybridized and covalently cross-linked to the outer surface of
the corresponding dendrimers, respectively. Excess capture and
fluorescent labeled oligonucleotides are then removed through
techniques such as size exclusion chromatography and density
gradient ultracentrifugation.
[0071] The concentration of dendrimer is determined by measuring
the optical density of the purified material at 260 nm on a UV/Vis
spectrometer. The fluorescence is measured at optimal signal/noise
wavelengths using a fluorometer (FluoroMax, SPEX Industries). Cy3
is excitable at 542 nm and the emission measured at 570 nm. Cy5 is
excitable at 641 nm and the emission at 676 nm.
[0072] In the preferred embodiments of the present invention, a
dendrimer is utilized having approximately 850 fluorescent dyes on
each molecule, such as the dendrimers available in the Genisphere,
Inc. 3DNA Array 900 labeling kit. Alternatively, prior dendrimers
having approximately three hundred dyes can be utilized, or
dendrimers having more than 500 fluorescent dyes.
[0073] The use of such dendrimer probes significantly increases
increasing sensitivity due to the dendrimers' superior signal
amplification capability. By increasing sensitivity, the amount of
RNA required for an assay is reduced, allowing the use of smaller
volumes of initial sample. In particular, the present invention can
be easily conducted using 0.25-1 microgram of total RNA, or, as low
as 100 nanograms of total RNA (0.1 micrograms); or with 1 to 1000
nanograms of poly A RNA (mRNA). Accordingly, a dendrimer having
approximately 850 or more fluorescent dyes, is preferably utilized
to obtain these improved results. As the sensitivity of detection
improves in the art (e.g. using improved dendrimers or other
improved labelling and signal molecules), smaller RNA samples may
be assayed using the present invention as well.
[0074] For the assay itself, a strategy is preferably used (a "two
step method") that employs successive hybridization steps where the
reverse transcribed cDNA is applied to the array for a sufficiently
long period to allow hybridization thereto, with hybridization of
the cDNA molecules to target immobilized probes being followed by a
washing procedure where the unbound cDNA and excess RT primer is
removed from the array. The cDNA preferably has a capture sequence
incorporated thereto for binding to a dendritic nucleic acid having
a label capable of generating a detectable signal, as disclosed
below. The fluorescently labeled dendrimer molecule (or another
molecule capable of binding to the capture sequence incorporated
into the cDNA) is subsequently applied to the washed array and
hybridizes to the cDNA associated capture sequence during this
second hybridization. Excess dendrimer is washed away during a
secondary washing procedure and the arrays are scanned to detect
signal generated by the label molecule.
[0075] If desired, in further preferred embodiments, temperature
cycling can be used to selectively control hybridization between
the target nucleic acid and the microarray, and hybridization
between the capture reagent and the microarray (preferably
cDNA--microarray hybridization and cDNA--dendrimer hybridization,
respectively). By using such cycling, hybridization can be
carefully controlled such that cDNA initially hybridizes only to
the microarray, with subsequent hybridization of cDNA to the
dendrimer. This procedure can be used to improve the kinetics of
hybridization of each of the two components, i.e. target nucleic
acid to probe, and capture reagent to target nucleic acid. Further
details regarding use of such temperature cycling are provided in
U.S. Nonprovisional application Ser. No. 10/050,088 filed on Jan.
14, 2001, U.S. Provisional Application No. 60/261,231 filed Jan.
13, 2001, and in published protocols by the present inventors and
by Genisphere, Inc. of Montvale, N.J., all of which are fully
incorporated herein by reference.
[0076] In the preferred embodiments of the present invention, the
nucleic acid synthesized will generally be DNA that has been
reverse transcribed from RNA derived from a naturally occurring
source, where the RNA may be selected from the group consisting of
total RNA, poly(A).sup.+ RNA, amplified RNA and the like. The
initial RNA source may be present in a variety of different
samples, where the sample will typically be derived from a
physiological source. The physiological source may be derived from
a variety of sources, with physiological sources of interest
including sources derived from single celled organisms such as
yeast or bacteria, and multicellular organisms, including plants
and animals, particularly mammals, where the physiological sources
from multicellular organisms may be derived from particular organs
or tissues of the multicellular organism, or from isolated cells
derived therefrom. In obtaining the sample RNAs to be analyzed from
the physiological source from which it is derived, the
physiological source may be subjected to a number of different
processing steps, where such known processing steps may include
tissue homogenation, cell isolation and cytoplasmic extraction,
nucleic acid extraction, poly A tailing, and the like. Methods of
isolating RNA from cells, tissues, organs or whole organisms are
known to those of ordinary skill in the art and are described, for
example, in Maniatis et al., Molecular Cloning: A Laboratory
Manual, 2.sup.nd ed., Cold Spring Harbor Laboratory Press, 1989,
and in Ausubel et al., Current Protocols in Molecular Biology, John
Wiley & Sons, Inc., 1998, both of which are fully incorporated
herein by reference.
[0077] The sample mRNA is preferably reverse transcribed into a
target nucleic acid in the form of a cDNA, by hybridizing an
oligo(dT) primer, or RT primer, to the mRNA under conditions
sufficient for enzymatic extension of the hybridized primer. The
primer will be sufficiently long to provide for efficient
hybridization to the mRNA tail, where the region will typically
range in length from 10 to 25 nucleotides, usually 10 to 20
nucleotides, and more usually from 12 to 18 nucleotides.
[0078] Recognizing that applications typically require the use of
sequence specific primers, the standard primers as used in the
present invention further include "capture sequence" nucleotide
portions. The preferred capture sequences referred to herein are
Cy3.RTM. RT primer capture sequences (Oligos etc., Inc,
Wilsonville, Oreg.) or Cy5.RTM. RT primer capture sequence (Oligos
etc., Inc, Wilsonville, Oreg.), as disclosed for example in
International Application No. PCT/US02/027799 filed 3 Sep. 2002,
which is fully incorporated herein by reference.
[0079] For custom primers, the capture sequences should be attached
to the 5' end of the corresponding custom oligonucleotide primer.
In this manner, the custom primer replaces the standard RT primer.
Since the present invention is devised for use with the standard RT
primer, some modifications may be required when substituting a
custom primer. Such modifications are known to those of ordinary
skill in the art and may include adjusting the amount and mixture
of primers based on the amount and type of RNA sample used. The
primer carries a capture sequence comprised of a specific sequence
of nucleotides, as described above. The capture sequence is
complementary to the oligonucleotides attached to the arms of
dendrimer probes which further carry at least one label. Such
complementary oligonucleotides may be acquired from any outside
vendor and may also be acquired as labeled moieties. The label may
be attached to one or more of the oligonucleotides attached to the
arms of the dendrimer probe, either directly or through a linking
group, as is known in the art. In the preferred embodiment, the
dendrimer probes are labeled by hybridizing and cross-linking
Cy3.RTM. or Cy5.RTM. labeled oligonucleotides to the dendrimer
arms. The Cy3.RTM. or Cy5.RTM. labeled oligonucleotides are
complementary to the Cy3.RTM. or Cy5.RTM. RT primer capture
sequences, respectively.
[0080] In generating the target nucleic acid sample, the primer is
contacted with the RNA in the presence of a reverse transcriptase
enzyme, and other reagents necessary for primer extension under
conditions sufficient for inducing first strand cDNA synthesis. A
variety of enzymes, usually DNA polymerases, possessing reverse
transcriptase activity can be used for the first strand cDNA
synthesis step. Examples of suitable DNA polymerases include the
DNA polymerases derived from organisms selected from the group
consisting of a thermophilic bacteria and archaebacteria,
retroviruses, yeasts, Neurosporas, Drosophilas, primates and
rodents. Suitable DNA polymerases possessing reverse transcriptase
activity may be isolated from an organism, obtained commercially or
obtained from cells which express high levels of cloned genes
encoding the polymerases by methods known to those of skill in the
art, where the particular manner of obtaining the polymerase will
be chosen based primarily on factors such as convenience, cost,
availability and the like. The order in which the reagents are
combined may be modified as desired.
[0081] In one preferred embodiment of the invention, the cDNA
synthesis protocol involves combining total RNA or poly(A).sup.+
RNA (mRNA), with RT primer and RNase free water to yield the RNA-RT
primer mix. In accordance with the present invention, very small
quantities of initial RNA can be utilized, as discussed below.
[0082] The RNA-RT primer mixture is then mixed and microfuged to
collect the contents at the bottom of the microfuge tube. The
RNA-RT primer mixture is then heated at a suitable temperature
(e.g. 80 degrees Celsius) for ten minutes and immediately
transferred to ice. In a separate microfuge tube on ice, RT buffer,
DTT (dithiothreitol), RNAse inhibitor, dNTP mix, and reverse
transcriptase enzyme are combined with RNase free water (the
Reaction Master Mix). This combination is gently mixed (but not
vortexed) and microfuged briefly to collect the contents at the
bottom of the microfuge tube to yield a reaction mixture. The
RNA-RT primer mixture is then mixed with the Reaction Master Mix
and incubated at a suitable temperature (e.g. about 42 degrees
Celsius) for a period of time sufficient for forming the first
strand cDNA primer extension product, which usually takes about 2
hours. The reaction is stopped using 1.0M NaOH/100 mM EDTA, and
then incubated at a suitable temperature (e.g. 65 degrees Celsius)
to denature the DNA/RNA hybrids and degrade the RNA. The reaction
is then neutralized with 2M Tris-HCl (pH 7.5).
[0083] Once this is completed, the cDNA is applied directly to the
array for hybridization thereto without post-synthesis
concentration of the cDNA. (In an additional embodiment, there is
no post-synthesis purification conducted of the cDNA, either).
Rather, the mixture obtained above is directly added to the
microarray and incubated at a second hybridization temperature and
for a sufficient time to allow the cDNA to bind to the microarray.
Suitable hybridization conditions are well known to those of skill
in the art and reviewed in Maniatis et al., supra, where conditions
may be modulated to achieve a desire specificity in hybridization.
If desired, a blocking LNA oligonucleotide can be used to reduce
non-specific binding between the cDNA and the array, as discussed
in International Application No. PCT/US02/027799 filed 3 Sep. 2002,
International Publication No. WO 03/020902 A2, which is fully
incorporated herein by reference. The array is then washed to
reduced background on the array (e.g. caused by free RT primer not
incorporated into cDNA molecules).
[0084] A label molecule (preferably a dendrimer carrying a desired
label) is then applied to the array for hybridization of the label
to the capture sequence of the cDNA to provide a detectable signal.
Following the hybridization step, a washing step is employed in
which unhybridized complexes are purged from the microarray, thus
leaving behind a visible, discrete pattern of hybridized
cDNA-dendrimer probes bound to the microarray. A variety of wash
solutions and protocols for their use are known to those of skill
in the art and may be used. The specific wash conditions employed
will necessarily depend on the specific nature of the signal
producing system that is employed, and will be known to those of
skill in the art familiar with the particular signal producing
system employed.
[0085] The resultant hybridization pattern of labeled cDNA
fragments may be visualized or detected in a variety of ways, with
the particular manner of detection being chosen based on the
particular label of the cDNA, where representative detection means
include scintillation counting, autoradiography, fluorescence
measurement, calorimetric measurement, light emission measurement
and the like.
[0086] Following hybridization and any washing step(s) and/or
subsequent treatments, as described above, the resultant
hybridization pattern is detected. In detecting or visualizing the
hybridization pattern, the intensity or signal value of the label
will be not only be detected but quantified, by which is meant that
the signal from each spot of the hybridization will be
measured.
[0087] Following detection or visualization, the hybridization
pattern can be used to determine quantitative and qualitative
information about the initial RNA sample. For example, information
can be obtained regarding the genetic profile of the labeled target
nucleic acid sample that was contacted with the microarray to
generate the hybridization pattern. From this data, one can also
derive information about the physiological source from which the
target nucleic acid sample was derived, such as the types of genes
expressed in the tissue or cell which is the physiological source,
as well as the levels of expression of each gene, particularly in
quantitative terms. Where one uses the subject methods in comparing
target nucleic acids from two or more physiological sources, the
hybridization patterns may be compared to identify differences
between the patterns. Where microarrays in which each of the
different probes corresponds to a known gene are employed, any
discrepancies can be related to a differential expression of a
particular gene in the physiological sources being compared. Thus,
for example, the subject methods find use in differential gene
expression assays, where one may use the subject methods in the
differential expression analysis of: diseased and normal tissue,
e.g. neoplastic and normal tissue, different tissue or subtissue
types; and the like.
[0088] Many other variations of the above procedures can be used
consistent with the present invention. For example, instead of
utilizing RNA extracted from a sample which is converted to cDNA
prior to hybridization, the present invention can be used with the
RNA sample directly. In one such embodiment, a suitable capture
sequence can be ligated to the RNA using known methods of splicing
RNA, such as through enzymatic means. Or, if the RNA includes a
specific oligonucleotide that is useful as a capture sequence, a
complementary oligonucleotide can be attached to a dendrimer to
label the RNA molecule. Further details regarding methods for
direct use of RNA without the need for reverse transcription are
provided in PCT Application No. PCT/US01/22818 filed Jul. 19, 2001,
which is fully incorporated herein by reference.
[0089] The invention is particularly suitable as for improved
synthesis and assay of cDNA using very small quantities of initial
starting materials, as the present invention allows the use of less
initial RNA sample than prior methods. For example, when partnered
with the 3DNA dendrimer technology (or other very sensitive signal
generating methods, e.g. relative light scatter detection using
nanogold labels, such as those of Genicon Inc./Invitrogen), this
cDNA method is particularly valuable as it allows for use of total
RNA samples down to 250 nanograms or less without any need for RNA
target amplification. The invention can be easily used with 0.25-1
microgram of sample, or, if desired with 100 nanograms of initial
total RNA sample (0.1 micrograms); or with 1 to 1000 nanograms of
poly A RNA (mRNA). This is about 2 orders of magnitude better than
direct incorporation methods, which require 20-50 micrograms of
total RNA, or 1 to 2.5 micrograms of poly A RNA (mRNA) for the same
level of sensitivity.
[0090] The present methods, therefore, allow for use of very small
starting samples that otherwise would be lost during the
post-synthesis concentration or purification steps. As the
sensitivity of detection increases in the art (e.g. using improved
dendrimers, or dendrimers having additional fluorescent dyes, or
other improved signal generating methods), smaller samples of RNA
can be utilized in conjunction with the present invention as well.
Those samples can likewise be applied to the microarray without
concentration of the cDNA post-synthesis.
[0091] The invention provides the ability to scale up the reaction
and yet still avoid the need for concentration of purification of
the final cDNA synthesis. A further benefit of the invention is the
reduction of non-specific background on microarrays caused directly
or indirectly by the presence of high amounts of cDNA, which are
avoided by using this method. Likewise, the method provides
increased data quality (specifically, the range of differentials in
a gene expression experiment), which is likely due to the very
small quantity of cDNA used in the array hybridization assay. This
is due to the fact that, as opposed to prior methods, in the
present invention no concentration is conducted of the cDNA post
synthesis. Previous methods generally require concentration of cDNA
for using the cDNA on most types of arrays due to the larger sample
size required with non-3DNA methods and the small volumes required
for the microarray hybridization.
[0092] Likewise, a further improvement provided herein is that the
present method does not require purification of the cDNA synthesis
reaction, which is normally performed for direct dye incorporation
cDNA synthesis to remove excess dye and enzyme not incorporated
into the cDNA.
[0093] In addition, the present invention provides the further
advantage of being readily automatable, as opposed to the
concentration and purification steps normally used in this context,
which are currently difficult to accomplish in an automated fashion
or robotically.
[0094] Further advantages, features and aspects of the present
invention will be apparent in conjunction with the following
examples.
Example 1
cDNA Synthesis from Total RNA
[0095] 1. In a microtube combine: [0096] 1-5 .mu.l total RNA
(0.25-1.0 g mammalian total RNA or 0.5-2.5 .mu.g plant total RNA)
[0097] 1 .mu.l RT Primer (5 pmole/.mu.l) [0098] Add Nuclease Free
Water to a final volume of 6 .mu.l [0099] This is the RNA-RT primer
mix. [0100] Note: the use of 5 pmole/.mu.l RT Primer may cause
non-specific background signal on certain types of arrays (i.e.
poly-L-lysine coated slides). This type of background may be
reduced by diluting the RT Primer by up to 2.5 fold with Nuclease
Free Water prior to its addition to the RNA sample (caution: avoid
diluting the primer to less than 2 pmole/.mu.l as this may cause
inefficient cDNA synthesis). [0101] 2. Mix and microfuge briefly to
collect contents in the bottom of the tube. [0102] 3. Heat to
80.degree. C. for five minutes and immediately transfer to ice for
2-3 minutes. [0103] 4. When provided as part of a kit, Reverse
transcriptase enzyme and the enzyme's reaction buffer can be
included within that kit or can be purchased separately. The use of
SuperScript IT Reverse Transcriptase Enzyme (Gibco Cat No.
18064-014-10,000 Units @ 200U/.mu.l) is recommended. [0104] The
Reaction Master Mix should be formulated to a final volume
dependent on the number of cDNA syntheses set up simultaneously.
Each cDNA synthesis requires 4.5 .mu.L of Reaction Master Mix. To
reduce pipetting errors, the Reaction Master Mix should contain at
least 9 .mu.L (enough for two cDNA syntheses).
[0105] Combine on ice in a separate microtube according to the
chart below: TABLE-US-00001 Number of cDNA synthesis 1 2 3 4 5 10.
. . 5X SuperScript II First Strand Buffer* 4.0 .mu.l 4.0 .mu.l 6.0
.mu.l 8.0 .mu.l 10.0 .mu.l 20.0 .mu.l dNTP mix 1.0 .mu.l 1.0 .mu.l
1.5 .mu.l 2.0 .mu.l 2.5 .mu.l 5.0 .mu.l 0.1 M DTT (supplied with
enzyme) 2.0 .mu.l 2.0 .mu.l 3.0 .mu.l 4.0 .mu.l 5.0 .mu.l 10.0
.mu.l Superscript II enzyme, 200 units 1.0 .mu.l 1.0 .mu.l 1.5
.mu.l 2.0 .mu.l 2.5 .mu.l 5.0 .mu.l Superase-in RNase Inhibitor 1.0
.mu.l 1.0 .mu.l 1.5 .mu.l 2.0 .mu.l 2.5 .mu.l 5.0 .mu.l Total
Volume 9.0 .mu.l 9.0 .mu.l 13.5 .mu.l 18.0 .mu.l 22.5 .mu.l 45.0
.mu.l
[0106] This is the Reaction Master Mix. (The Reaction Master Mix
should be kept on ice until used). [0107] 5. Gently mix (do not
vortex) and microfuge briefly to collect reaction mix contents in
the bottom of the tube. [0108] 6. Add 4.5 .mu.l of reaction mix
from step 5 to the 6 .mu.l of RNA-RT primer mix from step 1 (10.5
.mu.l final volume). [0109] 7. Gently mix (do not vortex) and
incubate at 42.degree. C. for two hours. [0110] 8. Stop the
reaction by adding 1.0 .mu.l of 1.0M NaOH/100 mM EDTA. [0111] 9.
Incubate at 65.degree. C. for ten minutes to denature the DNA/RNA
hybrids and degrade the RNA. [0112] 10. Neutralize the reaction
with 1.2 .mu.l of 2M Tris-HCl, pH 7.5. The cDNA synthesis
preparation is now ready for use in an experiment (microarrays,
blots, FISH, etc.), without any needed post-synthesis
concentration. If desired, the cDNA preparation can be used with
any post-synthesis purification as well.
Example 2
Method of cDNA Synthesis for Use with Microarrays without
Post-Synthesis Sample Concentration
[0113] The following method is an example of a method for synthesis
of cDNA for use with microarrays, wherein, in accordance with the
present invention, the method of synthesis avoids the need for
post-synthesis sample concentration. In the preferred embodiment
disclosed herein, the method is designed for use with reagents from
Genisphere, Inc., and in particular with the Genisphere.RTM. 3DNA
Array 900 kit (available from Genisphere, Inc. of Montvale, N.J.
and Hatfield Pa.), which is designed to provide increased
sensitivity on microarrays when using extremely small quantities of
RNA. (The instructions for use of the Array 900 kit provided with
the kit are fully incorporated herein by reference). Alternatively,
the method can be used with other kits or systems, using the method
for small volume synthesis disclosed below.
[0114] In the preferred method, better sensitivity is achieved
through the use of the modified cDNA synthesis protocol of the
present invention that eliminates post-synthesis sample
concentration, which avoids loss of sample during the concentration
process. Sensitivity can also be further enhanced by use of a
Genisphere.RTM. 3DNA Capture Reagent in the form of a dendrimer
containing approximately 850 fluorescent dyes and engineered for
more efficient hybridization kinetics, and by use of the
Genisphere.RTM. 2.times. Enhanced cDNA Hybridization Buffer
designed for better hybridization efficiency.
[0115] The Genisphere.RTM. 3DNA Array 900 kit is easy to use and is
designed for use with arrays printed with oligonucleotides or PCR
products (cDNA). First, either total or poly(A)+ RNA is reverse
transcribed, using a deoxynucleotide triphosphate mix and special
RT dT primer. Then, the cDNA and the fluorescent 3DNA reagent are
hybridized to the microarray in succession. The fluorescent 3DNA
reagent will hybridize to the cDNA because it includes a "capture
sequence" that is complementary to a sequence on the 5' end of the
RT primer.
[0116] The 3DNA Array 900 labeling system provides a more
predictable and consistent signal than direct or indirect dye
incorporation for two reasons. First, since the fluorescent dye is
part of the 3DNA reagent, it does not have to be incorporated
during the cDNA preparation. This avoids inefficient hybridization
of the cDNA to the array that results from the incorporation of
fluorescent dye nucleotide conjugates into the reverse transcript.
Second, because each 3DNA molecule contains an average of about 850
fluorescent dyes and each bound cDNA will be detected by a single
3DNA molecule, the signal generated from each message will be
largely independent of base composition or length of the
transcript. In contrast, the signal generated from each message
labeled through dye incorporation will vary depending on the base
composition or length of the message.
[0117] It should be noted that the array pattern produced by this
kit may differ somewhat from the pattern produced by direct or
indirect dye incorporation labeling methods when total RNA samples
are used. The reason for this is that reverse transcriptase enzyme
is known to label genomic DNA (without the need for a primer) as
well as RNA. Dye incorporation labeling methods can therefore
produce labeled genomic DNA. The labeled genomic DNA will bind to
microarrays, resulting in false positives for negative genes and/or
inappropriate and misleading fluorescence levels for array elements
simultaneously bound with cDNA produced by reverse transcription of
RNA. The 3DNA reverse transcription process, in contrast, utilizes
unlabeled nucleotides that cannot incorporate any fluorescence into
genomic DNA, thus eliminating the possibility of signal
contribution from genomic DNA. Because 3DNA labeling differs from
dye incorporation labeling in this way, the array pattern produced
may vary depending on which labeling method is used. However, in a
differential expression experiment, the expression patterns between
the two RNA samples should be the same regardless of the labeling
method, provided the genomic DNA has been eliminated from the
samples.
Kit Contents
(Certain components of the Genisphere.RTM. Array 900 Kit,
specifically Vials 1, 2 and 11, may not be compatible with other
microarray labeling kits).
[0118] Vial 1 Cy3/Alexa Fluor 546 (red cap) or Cy5/Alexa Fluor 647
(blue cap) 3DNA Array 900 Capture Reagent. (Dendrimer probe
reagents as described herein labeled with about 850 fluorescent
dyes per molecule. Dendrimers labeled with Cy3 (or Alexa Fluor 546)
contain the same unique capture sequence which targets the
dendrimer only to cDNAs synthesized with primers containing 5 prime
sequence complementary to the dendrimer bound capture sequence. Cy5
(or Alexa Fluor 647) labeled dendrimers contain a different capture
sequence, allowing differential binding of Cy3 and Cy5 labeled
dendrimers to cDNA). [0119] Vial 2 1.0 pmole/.mu.l RT Primer for
Cy3/Alexa Fluor 546 (red cap) or Cy5/Alexa Fluor 647 (blue cap).
(RT primers (48mers) comprising a 3 prime dT(17) and a 5 prime
31mer capture sequence). [0120] Vial 3 Deoxynucleotide Triphosphate
Mix (10 mM each dATP, dCTP, dGTP, and dTTP in water) [0121] Vial 4
Superase-In.TM. RNase inhibitor (Ambion) [0122] Vial 5 2.times.
Enhanced cDNA Hybridization Buffer (available from Genisphere Inc.
of Montvale, N.J. and Hatfield, Pa.) [0123] Vial 6
2.times.SDS-Based Hybridization Buffer (0.50M NaPO4; 1% SDS; 2 mM
EDTA; 2.times.SSC; 4.times.Denhardt's Solution) [0124] Vial 7
2.times. Formamide-Based Hybridization Buffer (50% Formamide;
8.times.SSC; 1% SDS; 4.times.Denhardt's Solution) [0125] Vial 8
Anti-Fade Reagent (0.1M DTT) [0126] Vial 9 LNA TM dT Blocker (for
use with PCR product (cDNA) microarrays) (a dT blocker comprising
36 nucleotides where 13 of the nucleotides are LNA (locked nucleic
acid, Exiqon AG) residues. See e.g., U.S. Nonprovisional
application Ser. No. 10/234,069 filed Sep. 3, 2002 which claims the
priority of U.S. Provisional Application Ser. No. 60/316,116 filed
Aug. 31, 2001, and "Methods for Blocking Nonspecific Hybridizations
of Nucleic Acids", International Application No. PCT/US02/027799
filed 3 Sep. 2002, International Publication No. WO 03/020902 A2,
all of which are fully incorporated herein by reference). [0127]
Vial 10 Nuclease Free Water (available from Ambion) [0128] Vial 11
5.0 pmole/.mu.l RT Primer for Cy3/Alexa Fluor 546 (red cap) or
Cy5/Alexa Fluor 647 (blue cap) [0129] (RT primers (48mers)
comprising a 3 prime dT(17) and a 5 prime 31mer capture sequence)
Vials 1-11 should be stored at -20.degree. C. in the dark. Vial 1
may be kept at 4.degree. C. for short-term storage (.about.1 week).
Other Materials Required Further materials required for use of this
method include: [0130] A microarray: commercial or in-house
prepared from either oligonucleotides or PCR/cDNA products [0131] A
microarray reader equipped to read Cy3/Alexa Fluor 546 and/or
Cy5/Alexa Fluor 647 fluorochromes [0132] Total RNA sample (greater
than or equal to 100 ng/.mu.l) or Poly(A).sup.+ RNA sample (greater
than or equal to 50 ng/.mu.l) [0133] Reverse Transcriptase enzyme
[0134] SuperScript II (Invitrogen Cat No. 18064-014-10,000 Units @
200 U/.mu.l) [0135] Genisphere RT Enzyme (Genisphere Cat No.
RT300320) [0136] or other equivalent reverse transcriptase enzyme
(Promega, etc). [0137] Cot-1 DNA (optional, species specific,
available from Invitrogen) [0138] Reagent Grade Deionized Water
(Recommended: VWR Cat No. RC9150-5) [0139] Note: As noted in the
Internet List Serve, MilliQ.RTM. water has been shown to damage Cy5
(http://groups.yahoo.com/group/microarray/messages/2867). [0140]
1.0M NaOH, 100 mM EDTA (cDNA synthesis stop solution) [0141] 2M
Tris-HCl, pH 7.5 [0142] 10 mM Tris-HCl, pH 8.0/1 mM EDTA
(1.times.TE Buffer) [0143] Glass coverslips (Corning brand
distributed by Fisher or VWR) or LifterSlipsO (Erie Scientific)
[0144] 2.times.SSC, 0.2% SDS buffer [0145] 2.times.SSC buffer
[0146] 0.2.times.SSC buffer [0147] Glass Coplin Jars (or
equivalent) [0148] 0.5M NaOH/50 mM EDTA (optional: for use with
Appendix A or B) [0149] 1M Tris-HCl, pH 7.5 (optional: for use with
Appendix A or B) [0150] Millipore Microcon.RTM.) YM-30 Centrifugal
Filter Device (30,000 molecular weight cutoff, Millipore Cat No.
42409) (optional: for use with Appendix C--Millipore Microcon
Procedure) [0151] Isopropanol (optional: for use with Appendix D)
[0152] 0.2% SDS buffer (optional: for use with Appendix D) [0153]
95% ethanol (optional: for use with Appendix D) [0154] DyeSaver
(Genisphere Cat No. Q100200) (optional: use to preserve fluorescent
signal and prevent photobleaching) RNA Preparation:
[0155] Preparation and use of high-quality RNA is critical to the
success of microarray experiments.
[0156] If degraded RNA is used, the RT reaction using dT primer
will only generate short poly dT tails as opposed to full length
cDNA, and little or no specific signal will be produced upon
subsequent array hybridization. If degraded RNA samples must be
used, good results can be obtained by labeling the samples with
Genisphere's 3DNA Array 350RP (Version 2) kit.
[0157] The use of an RNase inhibitor (Superase-In, Vial 4) is
strongly recommended. RNase inhibitor should be added to stored RNA
samples suspected of being contaminated with RNases. Inhibitor
should also be added during the reverse transcriptase reaction to
avoid RNA degradation during cDNA synthesis. Please refer to the
following references for more information regarding RNA degradation
by RNases (all of which are fully incorporated herein by
reference): Sambrook, J., Fritsch, E. F., and Maniatis, T.
Molecular Cloning, A Laboratory Manual (Second Edition) Cold Spring
Harbor Laboratory Press, 1989; Ausubel, F. M., Brent, R., Kingston,
R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K.
Current Protocols in Molecular Biology. John Wiley & Sons,
Inc., 1998.
[0158] The 3DNA Array 900 labeling system will not label genomic
DNA, so it is not essential to remove genomic DNA contamination.
However, the quantity and quality of the RNA present may be
determined more accurately if the genomic DNA is digested away.
Also, if the genomic DNA is allowed to remain in the sample, it may
bind to some of the RT enzyme and make the enzyme unavailable for
reverse transcription. RNase-free DNase is recommended for
degrading contaminating genomic DNA.
[0159] If DNase is used, it is essential that the DNase be
inactivated completely before proceeding with the cDNA synthesis
procedure to prevent degradation of the RT Primer. Methods for
inactivating DNase include phenol-chloroform extraction and use of
the RNeasy.RTM. kits from Qiagen. Inactivation of the DNase by high
temperature may not completely inactivate the enzyme.
[0160] High-quality RNA will have the following characteristics:
[0161] 1. OD 260/280 ratio will be between 1.9 and 2.1. [0162] 2.
On an agarose gel, total plant and mammalian RNA will be
represented as two sharp, bright bands. For mammalian RNA, the
bands will be at .about.4.5 kb and .about.1.9 kb, representing the
28S and 18S ribosomal sub-units, respectively. (Any suitable
protocol known in the art for RNA purification to produce such
high-quality RNA may be utilized, including, for example, those of
Genisphere.RTM.). Selection and Preparation of Microarrays:
[0163] Pre-spotted cDNA arrays manufactured by Genomic Solutions,
Agilent and Takara do not require special treatment prior to use.
Pre-spotted oligo arrays manufactured by MWG Biotech require
prewashing as described in Appendix E for optimal results. With
other purchased arrays, prepare or pre-treat the microarray as
described by the manufacturer. For arrays made "in-house", it is
recommended that one of the protocols in Appendix D be used for
pre-treating the arrays. These protocols do not require succinic
anhydride treatment and on many array types have yielded stronger
signal and lower background. (Please note: This protocol is not
compatible with Agilent arrays).
[0164] Genisphere recommends use of certain amino-silane coated
slides for spotting PCR products (cDNAs), particularly Clontech
DNA-Ready Type II Corning GAPS II and UltraGAPS, and Telechem
SuperAmine slides. These slides demonstrate good DNA binding when
used with 3DNA Array 900 kits.
[0165] Arrays prepared on poly-L-lysine, aldehyde or certain
amino-silane (e.g., Corning GAPS) surfaces may require either a
prewash or prehybridization step to reduce the background observed
after hybridization. Refer to Appendix E or F, respectively, for
procedures to help reduce background on microarrays.
[0166] As arrays age, they may exhibit lower specific signal and
higher levels of background noise. In some cases, as an array ages
the spotted probe demonstrates a "green" (Cy3) channel background.
This has been experienced with both commercial and "in-house"
arrays on all substrate surfaces. Quality control testing of both
commercial and "in-house" arrays should be performed immediately
after spotting (or receipt of arrays) and periodically thereafter
to establish non-specific background noise characteristics of the
arrays and other materials as they age. Also, all solutions used in
post-spotting array processing should be tested to assure
consistency and minimal contribution to non-specific array
background.
Hybridization Conditions:
[0167] Because microarrays vary, it is important to determine the
optimal hybridization conditions, including the optimal buffer
selection, for each array type. The Genisphere Array 900 kit, for
example, includes the following hybridization buffers: [0168] 1.
2.times. Enhanced cDNA Hybridization Buffer (Vial 5)--for use in
the cDNA hybridization step only, if additional sensitivity is
desired. This buffer may be used on arrays that tolerate higher
temperatures (up to 65.degree. C.). [0169] 2. 2.times.SDS-Based
Hybridization Buffer (Vial 6)--for use in both the cDNA and 3DNA
hybridization steps, this buffer may be used on arrays that
tolerate higher temperatures (up to 65.degree. C.). [0170] 3.
2.times. Formamide-Based Hybridization Buffer (Vial 7)--for use in
both the cDNA and 3DNA hybridization steps, this buffer is designed
for use at lower temperatures due to its higher stringency
formulation.
[0171] It is recommended that the hybridization buffers be tested
to determine which is best for the array type being utilized.
Please note that due to the viscous nature of the 2.times. Enhanced
cDNA Hybridization Buffer (Vial 5), a larger hybridization volume
may be required under a coverslip due to volume loss when
pipetting. Additionally, the ranges of hybridization temperatures
included in this product manual are provided as a guide. Genisphere
recommends that the optimal hybridization temperature be
empirically determined for each lot of microarrays. For example,
the poly-L-lysine surface of some arrays may begin to peel off at
the hybridization temperature required for use of the Vial 5 or 6
buffer. Use Vial 7 buffer as directed if you experience this
problem.
[0172] Because most PCR products contain poly(dA/dT) sequences, the
use of the LNA dT Blocker (Vial 9) is recommended on cDNA arrays.
The LNA dT Blocker is a high-performance poly T based blocking
reagent designed by Genisphere (patent pending, see, International
Application No. PCT/US02/027799 filed 3 Sep. 2002, International
Publication No. WO 03/020902 A2, which is fully incorporated herein
by reference). It is designed to completely block all poly A
sequences present in array features, including control spots
containing only poly dA sequences. This new blocking reagent
contains Locked Nucleic Acid (LNA) nucleotides (a patented
Exiqon.TM. technology) at key positions within the poly dT
synthetic strand. The presence of these modified nucleotides
stabilizes the hybridization between complementary strands of
nucleic acids, thus improving the blocking capacity of the poly dT
reagent. See reference 3 below for additional information relating
to LNA chemistry. Although average array signal intensity for a
blocked array may be lower compared to a non-blocked array,
specific signal from reversed transcribed cDNA binding to
complementary array elements should not be adversely affected.
While a volume of 2 .mu.l of the LNA dT Blocker (Vial 9) is
recommended for each hybridization, some arrays may demonstrate
better performance if additional LNA dT Blocker is used (3-4
.mu.l).
[0173] Add additional competitor nucleic acid as required (e.g.
species specific Cot-1 DNA from Invitrogen). Use competitor nucleic
acid at 1/10 by mass of input total RNA (i.e. use 100 ng of Cot-1
DNA for every microgram of total RNA). If too much competitor is
used, the signal may be reduced due to nonspecific interactions of
the excess competitor with the limited cDNA in the hybridization.
Denaturation of Cot-1 DNA and other competitor nucleic acids is
recommended (95-10.degree. C. for 5-10 minutes) prior to addition
to the hybridization mix.
Procedure for Use
[0174] The following summarizes the steps necessary to synthesize
cDNA from total RNA. If you are using poly(A).sup.+ RNA, follow the
procedure described in Appendix B for cDNA synthesis. Since
microarrays and RNA preparations vary in quality, the exact amount
of RNA required for a given experiment will typically range from
0.25-1.0 .mu.g of animal total RNA or 0.5-2.5 .mu.g of plant total
RNA. For new users, 1 .mu.g of animal total RNA or 2 .mu.g of plant
total RNA is recommended as a starting point for cDNA synthesis.
Larger or smaller amounts of RNA may be required to achieve optimal
results, depending on the quality of the RNA sample and the
array.
[0175] Larger quantities of RNA (>2 .mu.g) may be used for
large-scale cDNA synthesis. Appendix A details a simple procedure
for synthesizing cDNA from 2-50 .mu.g total RNA. Alternatively, the
procedure outlined on page 11 may be scaled up to accommodate
larger quantities of total RNA. However, the larger reaction volume
that results may require concentration of your cDNA samples (see
Appendix C). Also, cDNA prepared using procedures from certain
other Genisphere kits may also be used in conjunction with the
Array 900 kit. The following Genisphere kits contain components for
cDNA synthesis that generate cDNA compatible with the Array 900
3DNA Capture Reagents (Vial 1): [0176] Array 350 (Catalog No.
W300100, W300110, W300130, W300140, W300180, and W300184) [0177]
Array 350HS (Catalog No. H300100, H300110, H300130, H300140,
H300180, and H300184) [0178] Array 50 (Version 2) (Catalog No.
B100121, B100122, B100171, B100172, B100187, and B100189)
cDNA Synthesis from Total RNA
Method for Small Volume Synthesis without Post-Synthesis
Concentration
[0179] Please note: This procedure requires the pipetting of
extremely small volumes of samples and reagents. The use of a
pipetter designed for accurate and precise pipetting between 0.5
and 10 .mu.l is recommended. [0180] 1. In a 0.5 mL "non-stick"
microtube, prepare the RNA-RT primer mix: [0181] 1-5 .mu.l total
RNA (0.25-1.0 .mu.g mammalian total RNA or 0.5-2.5 .mu.g plant
total RNA) [0182] 1 .mu.l RT Primer (Vial 2 pmole/.mu.l) [0183] Add
Nuclease Free Water (Vial 10) to a final volume of 6 .mu.l. [0184]
2. Mix the RNA-RT primer mix and microfuge briefly to collect
contents in the bottom of the tube. [0185] 3. Heat to 80.degree. C.
for five minutes and immediately transfer to ice for 2-3 minutes.
Microfuge briefly to collect contents in the bottom of the tube and
return to ice.
[0186] 4. Prepare a Reaction Master Mix in a microtube on ice (see
table below). The Reaction Master Mix should be formulated to a
final volume dependent on the number of cDNA syntheses set up
simultaneously. Each cDNA synthesis requires 4.5 .mu.l of Reaction
Master Mix. To reduce pipetting errors, the Reaction Master Mix
should contain at least 9 .mu.l. TABLE-US-00002 Number of cDNA
syntheses 1 2 3 4 5 10 5X SuperScript II 4 .mu.l 6 .mu.l 8 .mu.l 10
.mu.l 12 .mu.l 22 .mu.l First Strand Buffer 0.1 M DTT (supplied 2
.mu.l 3 .mu.l 4 .mu.l 5 .mu.l 6 .mu.l 11 .mu.l with enzyme)
Superase-in RNAse 1 .mu.l 1.5 .mu.l 2 .mu.l 2.5 .mu.l 3 .mu.l 5.5
.mu.l Inhibitor (Vial 4) DNTP mix (Vial 3) 1 .mu.l 1.5 .mu.l 2
.mu.l 2.5 .mu.l 3 .mu.l 5.5 .mu.l Superscript II enzyme, 1 .mu.l
1.5 .mu.l 2 .mu.l 2.5 .mu.l 3 .mu.l 5.5 .mu.l 200 units Reaction
Master Mix 9 .mu.l 13.5 .mu.l 18 .mu.l 22.5 .mu.l 27 .mu.l 49.5
.mu.l Total Volume
Gently mix (do not vortex) and microfuge briefly to collect the
Reaction Master Mix contents in the bottom of the tube. Keep on ice
until ready to use. [0187] 5. Add 4.5 .mu.l of the Reaction Master
Mix from step 4 to the 6 .mu.l of RNA-RT primer mix from step 3
(10.5 .mu.l final volume). [0188] 6. Gently mix (do not vortex) and
incubate at 42.degree. C. for two hours. [0189] 7. Stop the
reaction by adding 1.0 .mu.l of 1.0M NaOH/100 mM EDTA. [0190] 8.
Incubate at 65.degree. C. for ten minutes to denature the DNA/RNA
hybrids and degrade the RNA. [0191] 9. Neutralize the reaction with
1.2 .mu.l of 2M Tris-HCl, pH 7.5. [0192] 10. Proceed to "Successive
Hybridization of cDNA and 3DNA to Microarray" below. Successive
Hybridization of cDNA and 3DNA to Microarray (Note: The following
protocol is not compatible with Agilent arrays). cDNA
Hybridization: [0193] 1. Thaw and resuspend the 2.times.
Hybridization Buffer (Vial 5, Vial 6, or Vial 7) by heating to
65-70.degree. C. for at least 10 minutes or until completely
resuspended. Vortex to ensure that the components are resuspended
evenly. If necessary, repeat heating and vortexing until all the
material has been resuspended. Microfuge for 1 minute. [0194] 2.
For each array, prepare the following cDNA Hybridization Mix for
use with a 24.times.50 glass coverslip*: [0195] 12.7 .mu.l cDNA
Synthesis #1 [0196] 12.7 .mu.l cDNA Synthesis #2 or Nuclease Free
Water (Vial 10) for single channel experiment [0197] 27.4 .mu.l
2.times. Hybridization Buffer (Vial 5, 6 or 7) (50% of the final
cDNA Hybridization Mix) [0198] 2 .mu.l LNA dT Blocker (Vial 9) (may
not be required for oligo arrays) [0199] 54.8 .mu.l total volume
*Alternatively, if a smaller coverslip and hybridization volume is
desired, simply prepare more cDNA than would be used on a single
array and load less cDNA volume per array. For example, a 30 .mu.l
hybridization volume containing cDNA from 500 ng of total RNA per
channel may be achieved by starting with 1000 ng of total RNA in
the cDNA synthesis and using one half the volume of the final cDNA
reverse transcription reaction. The final hybridization mix would
contain: [0200] 6.5 .mu.l cDNA synthesis #1 [0201] 6.5 .mu.l cDNA
synthesis #2 [0202] 15.0 .mu.l 2.times. Hybridization Buffer [0203]
2.0 .mu.l LNA Blocker [0204] 30.0 .mu.l total volume [0205]
Optional: 1.0 .mu.l C0T-1 DNA may also be added if desired (must be
denatured at 95-100.degree. C. for 10 minutes prior to use). [0206]
If a larger coverslip or LifterSlip is required, the cDNA
Hybridization Mix volume may be increased by adding equal volumes
of 2.times. Hybridization Buffer (Vial 5, 6 or 7) and Nuclease Free
Water (Vial 10). [0207] Note: Due to the viscous nature of the
2.times. Enhanced cDNA Hybridization Buffer (Vial 5), a larger
hybridization volume may be required due to volume loss when
pipetting. To address this, increase the total volume of the cDNA
Hybridization Mix by adding equal volumes of Nuclease Free Water
(Vial 10) and 2.times. Enhanced cDNA Hybridization Buffer (Vial 5).
[0208] (Note: This product has not been validated for use in
hybridization stations). [0209] 3. Gently vortex and briefly
microfuge the cDNA Hybridization Mix after addition of all
components. Incubate the Hybridization Mix first at 75-80.degree.
C. for 10 minutes, and then at the hybridization temperature until
loading the array (see the table located below step 5 for
recommended hybridization temperatures). Pre-warm the microarrays
to the hybridization temperature. [0210] 4. Gently vortex and
briefly microfuge the cDNA Hybridization Mix. Add the cDNA
Hybridization Mix to a pre-warmed microarray, taking care to leave
behind any precipitate at the bottom of the tube.
[0211] 5. Apply the appropriate glass coverslip to the array.
Incubate the array overnight in a dark humidified chamber at the
appropriate hybridization temperature: TABLE-US-00003 Spotted DNA
Size Vial 5 or 6 Buffer Vial 7 Buffer 30 mer 42-47.degree. C.
30-35.degree. C. 50 mer 55-60.degree. C. 42-48.degree. C. 70 mer
55-62.degree. C. 42-50.degree. C. PCR Products (cDNA) 55-65.degree.
C. 43-53.degree. C.
[0212] The hybridization temperatures recommended in this protocol
are intended as a starting point and should be used as a guide. It
may be necessary to adjust the temperatures to meet the stringency
requirements dictated by the nature of the nucleic acids spotted on
the array as well as the slide surface chemistry. In particular,
increasing the hybridization temperature by 5.degree. C. may remove
non-specific signal.
Post cDNA Hybridization Wash:
[0213] 1. Prewarm the 2.times.SSC, 0.2% SDS wash buffer: [0214]
55-65.degree. C. for PCR product (cDNA) arrays [0215] 42.degree. C.
for oligonucleotide spotted arrays [0216] 2. Remove the coverslip
by washing the array in prewarmed 2.times.SSC, 0.2% SDS for 2-5
minutes or until the coverslip floats off.* Additional time may be
required to remove the coverslip when the 2.times. Enhanced cDNA
Hybridization Buffer (Vial 5) is used. *Note: If the coverslip is
difficult to remove, this may be an indication of drying. To
prevent this problem from recurring in future experiments, increase
the total volume of the cDNA Hybridization Mix by adding equal
volumes of Nuclease Free Water (Vial 10) and 2.times. Hybridization
Buffer (Vial 5, 6 or 7). In addition, ensure that the hybridization
chamber is properly humidified and sealed. [0217] 3. Wash for 10-15
minutes in prewarmed 2.times.SSC, 0.2% SDS. [0218] 4. Wash for
10-15 minutes in 2.times.SSC at room temperature. [0219] 5. Wash
for 10-15 minutes in 0.2.times.SSC at room temperature. [0220] 6.
Transfer the array to a dry 50 mL centrifuge tube, orienting the
slide so that any label is down in the tube. Immediately centrifuge
without the tube cap for 2 minutes at 800-1000 RPM to dry the slide
(any delay in this step may result in high background). Avoid
contact with the array surface. [0221] 7. Further optimization of
wash conditions may be required to achieve optimal array
performance. If necessary to reduce background on the array, it is
recommended that the time of some or all of the washes be increased
to 15-20 minutes. Agitation during washing may also help to reduce
background due to non-specific binding to the surface of the array.
3DNA Hybridization: 1. Prepare the 3DNA Array 900 Capture Reagent
(Vial 1). It is necessary to break up aggregates that may form as a
result of the freezing process. [0222] a. Thaw the 3DNA Array 900
Capture Reagent (Vial 1) in the dark at room temperature for 20
minutes. [0223] b. Vortex at the maximum setting for 3 seconds and
microfuge briefly. [0224] c. Incubate at 50-55.degree. C. for 10
minutes. [0225] d. Vortex at the maximum setting for 3-5 seconds.
[0226] e. Microfuge the tube briefly to collect the contents at the
bottom. [0227] Be sure to check the sample for aggregates prior to
use and repeat vortex mixing if necessary. Aggregates may appear as
small air bubbles or flakes at the side of the tube below the
surface of the solution. Repeat steps a-e if necessary. 2. Thaw and
resuspend the 2.times. Hybridization Buffer (Vial 6 or Vial 7) by
heating to 70.degree. C. for at least 10 minutes or until
completely resuspended. Vortex to ensure that the components are
resuspended evenly. If necessary, repeat heating and vortexing
until all the material has been resuspended. Microfuge for 1
minute. [0228] Caution: Do not use the 2.times. Enhanced cDNA
Hybridization Buffer (Vial 5) in the 3DNA Hybridization step. 3.
The Anti-Fade Reagent (Vial 8) reduces fading of the fluorescent
dyes post hybridization. Prepare a stock solution by adding 111 of
Anti-Fade to 100 .mu.l of 2.times. Hybridization Buffer (Vial 6 or
Vial 7). Store any unused hybridization buffer at -20.degree. C.
and use within two weeks. However, do not use the Anti-Fade Reagent
if your arrays are printed on aldehyde-coated slides, as background
haze may result. Refreeze the Anti-Fade Reagent after use. 4. For
each array, prepare the following 3DNA Hybridization Mix for use
with a 24.times.50 glass coverslip*: [0229] 2.5 .mu.l Cy3/Alexa
Fluor S46 3DNA Array 900 Capture Reagent (Vial 1) [0230] 2.5 .mu.l
Cy5/Alexa Fluor 647 3DNA Array 900 Capture Reagent (Vial 1) [0231]
27.5 .mu.l 2.times. Hybridization Buffer (Vial 6 or 7) (50% of the
3DNA Hybridization Mix) [0232] 22.5 .mu.l Nuclease Free Water (Vial
10) [0233] 55.0 .mu.l total volume *Alternatively, if a smaller
coverslip and hybridization volume is desired, adjust the volume of
the 2.times. Hybridization Buffer (Vial 6 or 7) and Nunclease Free
Water (Vial 10) to a volume appropriate for the desired coverslip.
For example, the final 3DNA Hybridization Mix for a 30 .mu.l volume
would contain: [0234] 2.5 .mu.l Cy3/Alexa Fluor 546 3DNA Array 900
Capture Reagent (Vial 1) [0235] 2.5 .mu.l Cy5/Alexa Fluor 647 3DNA
Array 900 Capture Reagent (Vial 1) [0236] 15.0 .mu.l 2.times.
Hybridization Buffer (Vial 6 or 7) (50% of the 3DNA Hybridization
Mix) [0237] 10.0 .mu.l Nuclease Free Water (Vial 10) [0238] 30.0
.mu.l total volume [0239] Optional: 1.01 Cot-1 DNA may also be
added if desired (must be denatured at 95-100.degree. C. for 10
minutes prior to use). [0240] Note: For single channel expression
analysis, use 2.5 .mu.l of Nuclease Free Water (Vial 10) in place
of the second 3DNA Array 900 Capture Reagent. 5. Gently vortex and
briefly microfuge the 3DNA Hybridization Mix. Incubate the 3DNA
Hybridization Mix first at 75-80.degree. C. for 10 minutes, and
then at the hybridization temperature until loading the array (see
the table located below step 7 for recommended hybridization
temperatures). 6. Gently vortex and briefly microfuge the 3DNA
Hybridization Mix. Add the 3DNA Hybridization Mix to a pre-warmed
microarray, taking care to leave behind any precipitate at the
bottom of the tube. (Pre-warming the microarray to the
hybridization temperature may reduce background.)
[0241] 7. Apply a 24.times.50 glass coverslip to the array. If a
larger coverslip or Lifterslip is required, the 3DNA Hybridization
Mix volume may be increased by adding equal volumes of 2.times.
Hybridization Buffer (Vial 6 or 7) and Nuclease Free Water (Vial
10). Incubate the array for 4-5 hours in a dark humidified chamber
at the appropriate hybridization temperature: TABLE-US-00004
Spotted DNA Size Vial 6 Buffer Vial 7 Buffer 30 mer 55-65.degree.
C. 45-53.degree. C. 50 mer 55-65.degree. C. 45-53.degree. C. 70 mer
55-65.degree. C. 45-53.degree. C. PCR Products (cDNA) 60-65.degree.
C. 50-55.degree. C.
Post 3DNA Hybridization Wash: After hybridization the slides are
washed several times to remove unbound 3DNA molecules. Perform
these washes in the dark to avoid photobleaching and fading of the
fluorescent dyes. To reduce fading of Cy5 post hybridization, it
may also be beneficial to include DTT in the first two wash buffers
at a final concentration of 0.5-1 mM. Be sure to work with fresh
DTT, as old or poor quality DTT may cause an increase in background
visible as a "haze" in the Cy3 channel. Please refer to Appendix G
for recommendations for reducing the degradation of Cy5 when
performing microarray experiments. Caution: In the preparation of
wash buffers, avoid the use of water that may cause damage
Cy5/Alexa 647. As noted in the Internet List Serve, MilliQ.RTM.
water has been shown to damage Cy5
(http://groups.yahoo.com/group/microarray/messages/2867). Also, be
certain that any DEPC treated solutions have had all of the DEPC
fully removed (DEPC is a potent oxidizer). Alternatively, the use
of non-DEPC treated nuclease free solutions is recommended.
Commercially available solutions (water, buffers, etc.) from Ambion
have been found to work well with Cy5 labeled microarrays. In
addition to Ambion water, DI water from VWR (Cat. No. RC91505) is
also recommended. Water from Ambion and VWR have been validated for
use with microarrays and do not contain components that will
oxidize Cy5. [0242] 1. Prewarm the 2.times.SSC, 0.2% SDS wash
buffer as follows: [0243] 1. 65.degree. C. for PCR product (cDNA)
arrays and oligonucleotide arrays greater than 50 nucleotides long
[0244] 2. 42.degree. C. for oligonucleotide arrays less than 50
nucleotides long 8. Remove the coverslip by washing the array in
prewarmed 2.times.SSC, 0.2% SDS for 2-5 minutes or until the
coverslip floats off.* *Note: If the coverslip is difficult to
remove, this may be an indication of drying. To prevent this
problem from recurring in future experiments, increase the total
volume of the 3DNA Hybridization Mix by adding equal volumes of
Nuclease Free Water (Vial 10) and 2.times. Hybridization Buffer
(Vial 6 or 7). In addition, ensure that the hybridization chamber
is properly humidified and sealed. 9. Wash for 10-15 minutes in
prewarmed 2.times.SSC, 0.2% SDS. 10. Wash for 10-15 minutes in
2.times.SSC at room temperature. 11. Wash for 10-15 minutes in
0.2.times.SSC at room temperature. 12. Transfer the array to a dry
50 mL centrifuge tube, orienting the slide so that any label is
down in the tube. Immediately centrifuge without the tube cap for 2
minutes at 800-1000 RPM to dry the slide (any delay in this step
may result in high background). Avoid contact with the array
surface. Further optimization of wash conditions may be required to
achieve optimal array performance. If necessary to reduce
background on the array, increase the time of some or all of the
washes to 15-20 minutes. Agitation during washing may also help to
reduce background due to non-specific binding to the surface of the
array. Proceed to "Signal Detection" or first apply DyeSaver
coating (Genisphere Cat No. Q100200) to preserve fluorescent
signal. Signal Detection IMPORTANT: Store the array in the dark
until scanned. The fluorescence of the 3DNA reagents, especially
Cy5 and Alexa Fluor647, can diminish rapidly even in ambient light
because of oxidation. Please refer to Appendix F for
recommendations for reducing the degradation of Cy5/Alexa Fluor 647
when performing microarray experiments. Scan the microarray as
described by the scanner's manufacturer. Avoid excess multiple
scans as the dyes may photobleach from exposure to the scanner
light source. If using a Packard scanner, the recommendation is to
start by setting the laser at 80% power and either use the
"autobalance" feature or the table below to set up the initial
scanning parameters for proper channel balance. Adjustment of your
scanner laser power and photo-multiplier tube (PMT) voltage may be
required to balance the various fluorophore channels. If the PMT
setting is set too high, the background observed may be
unacceptable. In these insane the PMT setting should be reduced and
the laser power should be increased to optimize the signal-to-noise
ratio. However, to prevent photobleaching the fluorescent dyes,
especially Cy5/Alexa Fluor 647, after a single scan, avoid setting
the laser too high (>90-95% power).
[0245] Note: Balancing the image by offsetting the laser or PMT may
result in a non-linear distribution of the data between each
channel. In these instances, a statistical normalization may be
required. Please consult the instrument's user manual for further
instructions. TABLE-US-00005 Initial Scanner Setting for Packard
ScanArray 5000 4 Channel Scanner Dye Laser PMT Cy3/Alexa Fluor 546
80 70 +/- 5 Cy5/Alexa Fluor 647 80 65 +/- 5
[0246] If using an Axon 4000 series scanner, the recommended PMT
settings are as follows: TABLE-US-00006 Initial Scanner Setting for
Axon 4000B 2 Channel Scanner Dye Laser PMT Cy3/Alexa Fluor 546 100
500-700 volts Cy5/Alexa Fluor 647 100 600-800 volts
The following references provide further background as the
techniques discussed herein, and are fully incorporated herein by
reference: [0247] 1. Nilsen, T. W., Grayzel, J., and Prensky, W.
Dendritic Nucleic Acid Structures. J. Theor. Biol. (1997) 187:
273-284. [0248] 2. Stears, R. L., Getts, R. C., Gullans, S. R. A
novel, sensitive detection system for high-density microarrays
using dendrimer technology. Physiol Genomics 3: 93-99, 900. [0249]
3. Singh, S. K., Nielsen, P., Koshkin, A. A., and Wengel, J. LNA
(Locked Nucleic Acids): Synthesis and high-affinity nucleic acid
recognition. Chem. Commun., 455-456, 1998. Note: Cy is a trademark
of Amersham Biosciences; Alexa Fluor is a trademark of Molecular
Probes; RNeasy and Qiagen are trademarks of Qiagen Inc.;
Superase-In is a trademarked product of Ambion, Inc.; Exiqon and
LNA are trademarks of Exiqon A/S; Millipore, MilliQ and Microcon
are trademarks of Millipore, Inc.; LifterSlip is a trademark of
Erie Scientific Co.; and, 3DNA, Genisphere, Array 350RP, Array 350,
Array 50, Array 900 and DyeSaver are trademarks of Datascope Corp.
of Montvale, N.J.
APPENDIX A
[0249] Scaled-Up cDNA Preparation
A scaled up reverse transcription reaction can be performed from
2-50 .mu.g of total RNA to provide extra cDNA for duplicate
experiments, quantitation of the cDNA, or other parallel
analysis.
cDNA Synthesis:
[0250] 1. In a 0.5 or 1.5 mL RNase-free microcentrifuge tube,
prepare the RNA-RT primer mix: [0251] 1-10 .mu.l total RNA (2-50
.mu.g mammalian total RNA or 25-125 .mu.g plant total RNA) [0252] 1
.mu.l RT Primer (Vial 11, 5 pmole/.mu.l) [0253] Add RNase free
water to a final volume of 11 .mu.l [0254] 2. Mix the RNA-RT primer
mix and microfuge briefly to collect contents in the bottom of the
tube. [0255] 3. Heat to 80.degree. C. for ten minutes and
immediately transfer to ice for 2-3 minutes. [0256] 4. In a
separate microtube combine (on ice): [0257] 4 .mu.l 5.times.
Superscript II First Strand Buffer or equivalent reaction buffer
(supplied with enzyme) [0258] 2 .mu.l 0.1M dithiotreitol (DTT)
(supplied with the enzyme) [0259] 1 .mu.l Superase-In (Vial 4)
[0260] 1 .mu.l dNTP mix (Vial 3) [0261] 1 .mu.l Superscript II
enzyme, 200 Units
[0262] This is the reaction mix. The final volume should be 9
.mu.l. Gently mix (do not vortex) and microfuge briefly to collect
contents in the bottom of the tube. Keep on ice until used. [0263]
5. Add the 9 .mu.l of reaction mix from step 4 to the 11 .mu.l of
RNA-RT primer mix from step 3 (20 .mu.l final volume). [0264] 6.
Gently mix (do not vortex) and incubate at 42.degree. C. for two
hours. [0265] 7. Stop the reaction by adding 3.5 .mu.l of 0.5M
NaOH/50 mM EDTA. [0266] 8. Incubate at 65.degree. C. for ten
minutes to denature the DNA/RNA hybrids and degrade RNA. [0267] 9.
Neutralize the reaction with 5 .mu.l of 1M Tris-HCl, pH 7.5. The
resulting solution is your cDNA. [0268] 10. Dilute the cDNA to a
concentration appropriate for the desired hybridization volume.
[0269] Proceed to "Successive Hybridization of cDNA and 3DNA to
Microarray".
APPENDIX B
[0269] cDNA Preparation from Poly(A).sup.+ RNA
[0270] The procedure below summarizes the steps necessary to
synthesize cDNA from poly(A).sup.+ RNA. Since microarrays and RNA
preparations vary in quality, the exact amount of RNA required for
a given experiment will range from 12.5-50 ng of poly(A).sup.+ RNA.
For new users, 50 ng of poly(A).sup.+ RNA is recommended as a
starting point for cDNA synthesis.
cDNA Synthesis:
[0271] 1. In a 1.5 mL RNase-free microcentrifuge tube combine:
[0272] 1-10 .mu.l poly(A).sup.+ RNA (25-100 ng) [0273] 1 .mu.l RT
Primer (Vial 11, 5 pmole(.mu.l) [0274] Add RNase free water to a
final volume of 11 .mu.l [0275] This is the RNA-RT primer mix.
[0276] 2. Mix and microfuge briefly to collect contents in the
bottom of the tube. [0277] 3. Heat to 80.degree. C. for ten minutes
and immediately transfer to ice for 2-3 minutes. [0278] 4. In a
separate microtube combine (on ice): [0279] 4 .mu.l 5.times.
Superscript II First Strand Buffer or equivalent reaction buffer
(supplied with enzyme) [0280] 2 .mu.l 0.1M dithiotreitol (DTT)
(supplied with the enzyme) [0281] 1 .mu.l Superase-In (Vial 4)
[0282] 1 .mu.l dNTP mix (Vial 3) [0283] 1 .mu.l Superscript II
enzyme, 200 Units [0284] This is the reaction mix. The final volume
should be 9 .mu.l. Gently mix (do not vortex) and microfuge briefly
to collect contents in the bottom of the tube. Keep on ice until
used. [0285] 5. Add the 9 .mu.l of reaction mix from step 4 to the
11 .mu.l of RNA-RT primer mix from step 3 (20 .mu.l final volume).
[0286] 6. Gently mix (do not vortex) and incubate at 42.degree. C.
for two hours. [0287] 7. Stop the reaction by adding 3.5 .mu.l of
0.5M NaOH/50 mM EDTA. [0288] 8. Incubate at 65.degree. C. for ten
minutes to denature the DNA/RNA hybrids and degrade RNA. [0289] 9.
Neutralize the reaction with 5 .mu.l of 1M Tris-HCl, pH 7.5. The
resulting solution is your cDNA. [0290] 10. Dilute the cDNA to a
concentration appropriate for the desired hybridization volume.
[0291] Proceed to "Successive Hybridization of cDNA and 3DNA to
Microarray".
APPENDIX C
[0291] Concentration of cDNA
[0292] cDNA samples may be concentrated using the Millipore
Microcon YM-30 Centrifugal Filter Devices (30,000 molecular weight
cutoff, Millipore catalog number 42409). These devices are capable
of reducing the volume of the cDNA synthesis reaction to 3-10 .mu.l
in approximately 8-10 minutes. The procedure below reiterates the
manufacturer's directions with minor adaptations for the 3DNA Array
900 Kit.
Caution: Use of the Millipore Microcon YM-30 Centrifugal Filter
Devices may result in significant loss of small samples of cDNA
(<1.0 .mu.g).
Important: Users of the Microcon YM-30's should evaluate their own
centrifuge settings to determine the optimal time and speed
settings to yield final volumes of 3-10 .mu.l.
[0293] 1. Place the Microcon YM-30 sample reservoir into the 1.5 mL
collection tube. [0294] 2. Pre-wash the reservoir membrane by
adding 100 .mu.l 1.times.TE buffer to the Microcon YM-30 sample
reservoir. [0295] 3. Place the tube/sample reservoir assembly into
a fixed angle rotor tabletop centrifuge capable of 10-14,000 g.
[0296] 4. Spin for 3 minutes at 10-14,000 g. [0297] 5. Bring the
volume of the cDNA reaction to 100 .mu.l with 1.times.TE buffer.
Add all of the cDNA reaction to the Microcon YM-30 sample
reservoir. Do not touch the membrane with the pipet tip. [0298] 6.
Place the tube/sample reservoir assembly into a fixed angle rotor
tabletop centrifuge capable of 10-14,000 g. [0299] 7. Centrifuge
for 8-10 minutes at 10-14,000 g. [0300] 8. Remove the tube/sample
reservoir assembly. Separate the collection tube from the sample
reservoir with care, avoiding spilling any liquid in the sample
reservoir. [0301] 9. Add 5 .mu.l of 1.times.TE buffer to the sample
reservoir membrane without touching the membrane. Gently tap the
side of the concentrator to promote mixing of the concentrate with
the 1.times.TE buffer. [0302] 10. Carefully place the sample
reservoir upside down on a new collection tube. Centrifuge for 2
minutes at top speed in the same centrifuge. [0303] 11. Separate
the sample reservoir from the collection tube and discard the
reservoir. Note the volume collected in the bottom of the tube
(3-10 .mu.l total volume). The cDNA sample may be stored in the
collection tube for later use. [0304] 12. Add water to achieve the
desired volume of cDNA. Proceed to "Successive Hybridization of
cDNA and 3DNA to Microarray".
APPENDIX D
[0304] Array Processing Procedure (No Succinic Anhydride)
Option 1 (Recommended) (Cross-Link, Isopropanol Wash, and
Boil):
[0305] 1. Preheat 2 liters of reagent grade deionized distilled
water (best quality water available) to 95.degree. C.-100.degree.
C. (boiling) in a 4 liter beaker on a hot plate. [0306] 2. Transfer
250 mL of isopropanol into a glass rectangular staining dish and
place a small stir bar into the dish. Place the dish on a magnetic
stir plate and allow the bar to stir at a slow steady rate. [0307]
3. Retrieve the unprocessed arrays. Carefully, pick up one slide by
the corner and hold it in the steam above the boiling water (from
step 1) for five seconds. Make sure the arrays are facing up. Wave
the slide in the air for three seconds and place onto a fiber free
lab wipe, array side up. Repeat until eight slides have been
hydrated and dried [0308] 4. Transfer the eight slides, array side
up, to a cross-linker set to 50-220 mJ. [0309] 5. After
cross-linking, transfer the eight arrays into a glass/metal Wheaton
staining slide holder with grooves (do not place slides on each end
groove). Put the holder with the slides into the isopropanol (from
step 2) and incubate for 15 minutes with stirring. [0310] 6.
Transfer the slide holder to the boiling water (from step 1) and
incubate for 8-10 minutes. Make sure the slides are under the
water. [0311] 7. Remove the slide holder from the boiling water and
place onto a lab wipe to remove excess liquid. The arrays are now
ready for hybridization. Option 2 (Cross-Link, SDS Wash, Boil, and
Cold Ethanol Rinse): [0312] 1. Prepare 2 liters of a 0.2% SDS
solution in reagent grade deionized distilled water (best quality
water). For example, mix 40 mL of 10% SDS and 1960 mL of water in a
two liter autoclaved glass bottle. Filter the solution to remove
any precipitated SDS. Transfer 250 mL of this 0.2% SDS solution
into a glass rectangular staining dish and place a small stir bar
into the dish. Place the dish on a magnetic stir plate and allow
the bar to stir at a slow steady rate. [0313] 2. Preheat 2 liters
of reagent grade deionized distilled water (best quality water
available) to 95.degree. C.-100.degree. C. (boiling) in a 4 liter
beaker on a hot plate. [0314] 3. Transfer 2 liters of reagent grade
deionized distilled water to a 4 liter beaker. Keep at room
temperature. [0315] 4. Transfer 250 mL of ethanol into a glass
rectangular staining dish. Place this dish into an ice bucket to
set up an ice cold ethanol bath. [0316] 5. Retrieve the unprocessed
arrays. Carefully, pick up one slide by the corner and hold it in
the steam above the boiling water (from step 2) for five seconds.
Make sure the arrays are facing up. Wave the slide in the air for
three seconds and place onto a fiber free lab wipe, array side up.
Repeat until eight slides have been hydrated and dried. [0317] 6.
Transfer the eight slides, array side up, to a cross-linker set to
50-220 mJ. [0318] 7. After cross-linking, transfer the eight arrays
into a glass/metal Wheaton staining slide holder with grooves (do
not place slides on each end groove). Put the holder with the
slides into the 0.2% SDS (from step 1) and incubate for 10 minutes
with stirring. [0319] 8. Remove the slide holder and place onto a
lab wipe to remove excess liquid. Then dunk the holder into the 2
liters of room temperature water (from step 3) five times. [0320]
9. Transfer the slide holder to the boiling water (from step 2) and
incubate for 8-10 minutes. Make sure the slides are under the
water. [0321] 10. Remove the slide holder from the boiling water
and place onto a lab wipe to remove excess liquid. Transfer the
slide holder into the ice cold ethanol (from step 4) and incubate
for five minutes. Make sure the slides are under the ethanol.
[0322] 11. Remove the slide holder and place onto a lab wipe to
remove excess liquid. Transfer each slide into a 50 mL centrifuge
tube. Centrifuge at 1000 rpm for 3 minutes to dry the slides. The
arrays are now ready for hybridization.
APPENDIX E
[0322] Array Prewashing Procedure to Reduce Background
[0323] 1. Wash the microarray by the following conditions: [0324]
a. 2.times.SSC/0.2% SDS for 20 minutes at 55.degree. C. [0325] b.
0.2.times.SSC for 5 minutes at room temperature [0326] c. deionized
distilled water for 3 minutes at room temperature [0327] 2.
Immediately transfer the array to a dry 50 mL centrifuge tube. Do
this quickly to avoid streaky background on the slide. Orient the
slide so that any label is down in the tube. Centrifuge without the
tube cap for 2 minutes at 800-1000 RPM to dry the slide. Avoid
contact with the array surface. Transfer the microarray array to
dish or Coplin jar with 0.2.times.SSC at room temperature for 5
minutes. The array is now ready for either prehybridization or
hybridization with cDNA.
APPENDIX F
[0327] Array Prehybridization to Reduce Background:
Non-specific binding to the array surface is a common problem on
many array types. The prehybridization protocol described below is
recommended for reducing some types of non-specific binding,
thereby reducing the background seen post-hybridization.
1. Prewarm the microarray to 50.degree. C. for 10 minutes.
[0328] 2. Thaw and resuspend the 2.times. Hybridization Buffer
(Vial 7) by heating to 70.degree. C. for at least 10 minutes or
until completely resuspended. Vortex to ensure that the components
are resuspended evenly. If necessary, repeat heating and vortexing
until all the material has been resuspended. Microfuge for 1
minute.
3. Prepare Prehybridization Mix as follows:
[0329] 25 .mu.l 2.times. Formamide-Based Hybridization Buffer (Vial
7) [0330] 1 .mu.l Human Cot-1 DNA [0331] 24 .mu.l Nuclease free
water 4. Heat the Prehybridization Mix to 80.degree. C. for 10
minutes. 5. Apply the Prehybridization Mix to the prewarmed
microarray and cover with a 24.times.60 mm coverslip. 6. Incubate
at 50.degree. C. for 1-2 hours. 7. Wash the array by the following
conditions: [0332] a. 2.times.SSC, 0.2% SDS for 15 min at
60-65.degree. C. [0333] b. 2.times.SSC for 10 min at room
temperature. [0334] c. 0.2.times.SSC for 10 min at room
temperature. 8. Immediately transfer the array to a dry 50 mL
centrifuge tube. Do this quickly to avoid streaky background on the
slide. Orient the slide so that any label is down in the tube.
Centrifuge without the tube cap for 2 minutes at 800-1000 RPM to
dry the slide. Avoid contact with the array surface.
[0335] The array is now ready for hybridization with cDNA.
APPENDIX G
Recommendations for Reducing the Degradation of Cy5 or Alexa Fluor
647 when Performing Microarray Experiments
[0336] Cy5/Alexa Fluor 647 dye performance may be affected by a
variety of factors that are particularly prevalent during the
summer months. Exposure of the Cy5/Alexa Fluor 647 dye solutions
and the hybridized arrays to light and to oxidative environments
may cause rapid fading of the Cy5/Alexa Fluor 647 dye, regardless
of the labeling system used. Limiting or controlling the exposure
of the arrays to these environments has been shown to significantly
reduce Cy5/Alexa Fluor 647 fading.
Below are recommendations for reducing the degradation of Cy5/Alexa
Fluor 647 when performing microarray experiments:
1. Always keep solutions and arrays containing Cy5/Alexa Fluor 647
away from light, particularly sunlight Cy5/Alexa Fluor 647 will
photobleach when exposed to light, including normal fluorescent
lighting.
[0337] 2. Protect the hybridized, dried array from contact with
air, particularly on hot and sunny days. Ambient ozone levels
resulting from summertime air pollution can cause oxidative
degradation Cy5/Alexa Fluor 647. Keeping the Cy5/Alexa Fluor
647-containing arrays in an inert atmosphere (nitrogen) in a small
container (50mL tube) can significantly delay fading of the
Cy5/Alexa Fluor 647. Some investigators also add small quantities
of DTT or beta mercapto-ethanol (BME) to the bottom of the tube to
further promote a reducing micro-environment (be certain to avoid
contact of the array with these chemicals).
3. Use the Anti-Fade Reagent (provided with the 3DNA kits) in the
hybridization solution containing Cy5/Alexa Fluor 647 Capture
Reagent. The Anti-Fade Reagent has anti-oxidant properties that
will retard the oxidative process.
[0338] 4. In the preparation of wash buffers, avoid the use of
water that may cause damage Cy5/Alexa 647. As noted in the Internet
List Serve, MilliQ.RTM. water has been shown to damage Cy5
(http://groups.yahoo.com/group/microarray/messages/2867). Also, be
certain that any DEPC treated solutions have had all of the DEPC
fully removed (DEPC is a potent oxidizer). Alternatively, the use
of non-DEPC treated nuclease free solutions is recommended.
Commercially available solutions (water, buffers, etc.) from Ambion
have been found to work well with Cy5 labeled microarrays. In
addition to Ambion water, DI water from VWR (Cat. No. RC91505) is
also recommended. Water from Ambion and VWR have been validated for
use with microarrays and do not contain components that will
oxidize Cy5.
[0339] 5. Add a small quantity of dithiothreotol (DTT) to the post
hybridization wash buffers, i.e. 0.1 mM final concentration. This
potent reducing agent will protect the Cy5/Alexa Fluor 647 on the
array from attack by any oxidative agents in the wash buffers.
6. Always be certain to mix your 3DNA Cy3/Alexa Fluor 546 and
Cy5/Alexa Fluor 647 Capture Reagents (Vial 1) to break up any
aggregates that may form during storage:
[0340] a. Thaw the 3DNA Capture Reagent (Vial 1) in the dark at
room temperature for 20 minutes. [0341] b. Vortex at the maximum
setting for 3 seconds and microfuge briefly (1 second). [0342] c.
Incubate at 50-55.degree. C. for 10 minutes. [0343] d. Vortex at
the maximum setting for 3-5 seconds. [0344] e. Microfuge the tube
briefly to collect the contents at the bottom. [0345] Be sure to
check the sample for aggregates prior to use and repeat vortex
mixing if necessary. Aggregates may appear as small air bubbles or
flakes at the side of the tube below the surface of the solution.
Repeat steps a-e if necessary. 7. If the above recommendations do
not eliminate Cy5 degradation problems, the likely cause is
exposure to atmospheric pollutants. To address this issue,
Genisphere has developed a reagent, DyeSaver (Genisphere cat. no.
Q100200), that is applied to the array after the final wash and
spin. DyeSaver is easy to use, compatible with most array surface
chemistries, and protects Cy5 from atmospheric oxidation for up to
three weeks. DyeSaver has also been shown to reduce Cy5 damage due
to photobleaching.
[0346] Having described this invention with regard to specific
embodiments, it is to be understood that the description is not
meant as a limitation since further embodiments, modifications and
variations may be apparent or may suggest themselves to those
skilled in the art. It is intended that the present application
cover all such embodiments, modifications and variations.
Sequence CWU 1
1
2 1 12 RNA Unknown Seq. ID. No. 1 is a Eukaryotic Poly A tail.
("Unknown" chosen, since sequence is not an Artificial Sequence,
and is not meant to represent genetic material from just one
Genus/Species, but rather is common sequence among eukaryotes.)
polyAsite (1)..(12) 1 aaaaaaaaaa aa 12 2 12 DNA Artificial Sequence
Chemically synthesized (Poly T for hybridization to Poly A tail) 2
tttttttttt tt 12
* * * * *
References